Vesicle membrane proteins

ABSTRACT

The invention provides mammalian cDNAs which encode mammalian vesicle membrane proteins. It also provides for the use of the cDNAs, fragments, complements, and variants thereof and of the encoded proteins, portions thereof and antibodies thereto for diagnosis and treatment of cell proliferative disorders, particularly cancers of the colon, breast, ovary, uterus, prostate, adrenal gland, and thyroid, and thyroid follicular adenoma and thyroid lymphocytic thyroiditis. The invention additionally provides expression vectors and host cells for the production of the proteins and transgenic model systems.

[0001] This application is divisional application of U.S. Ser. No.09/718,996 filed Nov. 22, 2000, which is a continuation-in-part of U.S.Ser. No. 08/959,004, filed Oct. 28, 1997, now U.S. Pat. No. 6,197,543.

FIELD OF THE INVENTION

[0002] This invention relates to mammalian cDNAs which encode vesiclemembrane proteins and to the use of the cDNAs and the encoded protein inthe diagnosis and treatment of cell proliferative disorders,particularly cancers of the colon, breast, ovary, uterus, prostate,adrenal gland, and thyroid, and thyroid follicular adenoma and thyroidlymphocytic thyroiditis.

BACKGROUND OF THE INVENTION

[0003] Phylogenetic relationships among organisms have been demonstratedmany times, and studies from a diversity of prokaryotic and eukaryoticorganisms suggest a more or less gradual evolution of molecules,biochemical and physiological mechanisms, and metabolic pathways.Despite different evolutionary pressures, the proteins of nematode, fly,rat, and man have common chemical and structural features and generallyperform the same cellular function. Comparisons of the nucleic acid andprotein sequences from organisms where structure and/or function areknown accelerate the investigation of human sequences and allow thedevelopment of model systems for testing diagnostic and therapeuticagents for human conditions, diseases, and disorders.

[0004] Eukaryotic organisms are distinct from prokaryotes in possessingmany intracellular organelle structures. Many of the metabolic reactionswhich separate eukaryotic biochemistry from prokaryotic biochemistrytake place within these structures. In particular, many cellularfunctions require very strict reagent conditions, and the organellesenable compartmentalization and isolation of reactions which mightotherwise cripple cytosolic metabolic processes.

[0005] Isolation of intracellular organelles from rat liver hasdemonstrated the presence of two distinct organelles, the lysosome andthe peroxisome (de Duve (1996) Ann NY Acad Sci 804:1-10). Lysosomes arethe site of degradation of obsolete intracellular material duringautophagy and of extracellular molecules following endocytosis andphagocytosis. They are derived from endosomes, which in turn are formedfrom budding of the trans-Golgi network (TGN) or from clathrin-coatedmembrane vesicles invaginating from the plasma membrane. Lysosomescontain hydrolytic enzymes, and the enveloping membranes of lysosomesand early/late endosomes are enriched in highly glycosylatedtransmembrane proteins of largely unknown function. Some lysosomalmembrane proteins follow the constitutive secretory pathway and reachlysosomes indirectly via the cell surface. Other membrane proteins exitthe TGN in clathrin-coated vesicles for direct delivery to endosomes andto lysosomes (Hunziker and Geuze (1996) BioEssays 18:379-389).

[0006] Genetic studies in yeast and biochemical studies in animal cellshave provided evidence that the endocytic pathways and protein sortingin all eukaryotes probably share common enzymes and membrane components.An endocytic endosomal intermediate is responsible for the transport ofthe pheromone alpha-factor from the plasma membrane to the vacuole ofthe yeast, Saccharomyces cerevisiae. Proteins of the yeast endosomalmembrane which may contribute to the transport of alpha-factor have beeninvestigated in some detail. In particular, a protein with ten potentialtransmembrane domains, the EMP70 (p24a) precursor, has been identified(Singer-Kruger et al. (1993) J Biol Chem 268:14376-14386). Electronmicroscopic examination of yeast cells lacking functional EMP70 (p24a)shows a decrease in steady state vesicle accumulation and this suggeststhat EMP70 (p24a) is necessary for efficient vesicle budding (Stamnes etal. (1995) Proc Natl Acad Sci 92:8011-8015). A similar protein,KIAA0255, has been identified in a human myoblast cell line (Nagase etal. (1996) DNA Res 3:321-329).

[0007] Protein sorting by transport vesicles, such as the endosome, hasimportant consequences for a variety of physiological processesincluding cell growth, the biogenesis of distinct intracellularorganelles, endocytosis, and the controlled release of hormones andneurotransmitters (Rothman and Wieland (1996) Science 272:227-234). Inparticular, neurodegenerative disorders and other neuronal pathologiesare associated with biochemical flaws during endosomal protein sortingor endosomal biogenesis (Mayer et al. (1996) Adv Exp Med Biol389:261-269).

[0008] The peroxisome is the site of many important metabolic reactionsin eukaryotes such as lipid metabolism and gluconeogenesis, and isthought to cooperate intimately in biochemical reactions with thechloroplast (in plants and some protists) and the mitochondrion (inprotists, animals, and plants). Peroxisomes are independent organellesand are not members of the secretory pathway family of organelles. Theyare characterized by a single membrane and a finely granulated matrixand are the site of many peroxide-generating oxidative reactions in thecell. Peroxisomes are unique among eukaryotic organelles in that theirsize, number, and enzyme content vary depending upon organism, celltype, and metabolic needs. Assembly of peroxisomes and their contentswithin the cell is termed biogenesis. Perixosome biogenesis can bedivided into the following specific tasks: (1) membrane lipidacquisition, (2) proliferation/replication, (3) segregation, and (4)protein import. The majority of peroxisome-associated proteins aremembrane-bound or are found proximal to the cytosolic or the lumenalside of the peroxisome membrane (Waterham and Cregg (1996) BioEssays19:57-66).

[0009] Genetic defects in peroxisome proteins which result inperoxisomal deficiencies have been linked to a number of humanpathologies, including Zellweger syndrome, rhizomelic chonrodysplasiapunctata, X-linked adrenoleukodystrophy, acyl-CoA oxidase deficiency,bifunctional enzyme deficiency, classical Refsum's disease, DHAP alkyltransferase deficiency, and acatalasemia (Moser and Moser (1996) Ann NYAcad Sci 804:427-441). Some of these peroxisome proteins are requiredfor intracellular assembly of the organelle, including PAF-1, PXR1, andPXAAA1 (Dodt et al. (1996) Ann NY Acad Sci 804:516-523). Membraneprotein homologs and their cDNA counterparts have been isolated frommany organisms including the cyanobacterium Synechocystis (s11621),Candida boidinii (PMP20), and rat (peroxisomal 22 kDa membrane protein,PMP22) (Kaneko et al. (1996) DNA Res 3:109-136; Garrard and Goodman(1989) J Biol Chem 264:13929-13937; and Kaldi et al. (1993) FEBS Lett315:217-222). An mRNA which has some homology with peroxisome membraneproteins is downregulated in adenovirus 5-infected HeLa cells (DRAV5;Tomilin and Doerfler (1997) GenBank g1773069). Peroxisomal membraneproteins isolated from human liver include two integral membraneproteins of 22 kDa and 17 kDa (Santos et al. (1994) J Biol Chem269:24890-24896). In addition, Gartner et al. (1991; Pediatr Res29:141-146) found a 22 kDa integral membrane protein associated withlower density peroxisome-like subcellular fractions in patients withZellweger syndrome.

[0010] The discovery of mammalian cDNAs encoding vesicle membraneproteins satisfies a need in the art by providing compositions which areuseful in the diagnosis and treatment of cell proliferative disorders,particularly cancers of the colon, breast, ovary, uterus, prostate,adrenal gland, and thyroid, and thyroid follicular adenoma and thyroidlymphocytic thyroiditis.

SUMMARY OF THE INVENTION

[0011] The invention is based on the discovery of mammalian cDNAs whichencode vesicle membrane proteins, VMP, which are useful in the diagnosisand treatment of cell proliferative disorders, particularly cancers ofthe colon, breast, ovary, uterus, prostate, adrenal gland, and thyroid,and thyroid follicular adenoma and thyroid lymphocytic thyroiditis.

[0012] The invention provides an isolated mammalian cDNA or a fragmentthereof encoding a mammalian protein or a portion thereof selected fromthe group consisting of the amino acid sequences of SEQ ID NO:1 and SEQID NO:2, a variant having at least 80% identity to the amino acidsequences of SEQ ID NO:1 or SEQ ID NO:2, an antigenic epitope of SEQ IDNO:1 or SEQ ID NO:2, an oligopeptide of SEQ ID NO:1 or SEQ ID NO:2, anda biologically active portion of SEQ ID NO:1 or SEQ ID NO:2.

[0013] The invention also provides an isolated mammalian cDNA or thecomplement thereof selected from the group consisting of a nucleic acidsequence of SEQ ID NO:3 and SEQ ID NO:26, a variant having at least 80%identity to the nucleic acid sequences of SEQ ID NO:3 or SEQ ID NO:26, afragment of SEQ ID NO:3 comprising SEQ ID NOs:4-10 or a fragment of SEQID NO:26 comprising SEQ ID NOs:27-37, and an oligonucleotide of SEQ IDNOs:3-50. The invention additionally provides a composition, asubstrate, and a probe comprising the cDNA, or the complement of thecDNA, encoding VMP. The invention further provides a vector containingthe cDNA, a host cell containing the vector and a method for using thecDNA to make VMP. The invention still further provides a transgenic cellline or organism comprising the vector containing the cDNA encoding VMP.The invention additionally provides a mammalian fragment or thecomplement thereof selected from the group consisting of SEQ IDNOs:11-25 and 38-50. In one aspect, the invention provides a substratecontaining at least one of these fragments. In a second aspect, theinvention provides a probe comprising the fragment which can be used inmethods of detection, screening, and purification. In a further aspect,the probe is a single stranded complementary RNA or DNA molecule.

[0014] The invention provides a method for using a cDNA to detect thedifferential expression of a nucleic acid in a sample comprisinghybridizing a probe to the nucleic acids, thereby forming hybridizationcomplexes and comparing hybridization complex formation with a standard,wherein the comparison indicates the differential expression of the cDNAin the sample. In one aspect, the method of detection further comprisesamplifying the nucleic acids of the sample prior to hybridization. Inanother aspect, the method showing differential expression of the cDNAis used to diagnose cell proliferative disorders, particularly cancersof the colon, breast, ovary, uterus, prostate, adrenal gland, andthyroid, and thyroid follicular adenoma and thyroid lymphocyticthyroiditis.

[0015] The invention additionally provides a method for using a cDNA ora fragment or a complement thereof to screen a library or plurality ofmolecules or compounds to identify at least one ligand whichspecifically binds the cDNA, the method comprising combining the cDNAwith the molecules or compounds under conditions allowing specificbinding, and detecting specific binding to the cDNA, thereby identifyinga ligand which specifically binds the cDNA. In one aspect, the moleculesor compounds are selected from aptamers, DNA molecules, RNA molecules,peptide nucleic acids, artificial chromosome constructions, peptides,transcription factors, repressors, and regulatory molecules.

[0016] The invention provides a purified mammalian protein or a portionthereof selected from the group consisting of the amino acid sequencesof SEQ ID NO:1 or SEQ ID NO:2, a variant having at least 85% identity tothe amino acid sequences of SEQ ID NO:1 or SEQ ID NO:2, an antigenicepitope of SEQ ID NO:1 or SEQ ID NO:2, an oligopeptide of SEQ ID NO:1 orSEQ ID NO:2, and a biologically active portion of SEQ ID NO:1 or SEQ IDNO:2. The invention also provides a composition comprising the purifiedprotein or a portion thereof in conjunction with a pharmaceuticalcarrier. The invention further provides a method of using VMP to treat asubject with cell proliferative disorders, particularly cancers of thecolon, breast, ovary, uterus, prostate, adrenal gland, and thyroid, andthyroid follicular adenoma and thyroid lymphocytic thyroiditiscomprising administering to a patient in need of such treatment thecomposition containing the purified protein. The invention still furtherprovides a method for using a protein to screen a library or a pluralityof molecules or compounds to identify at least one ligand, the methodcomprising combining the protein with the molecules or compounds underconditions to allow specific binding and detecting specific binding,thereby identifying a ligand which specifically binds the protein. Inone aspect, the molecules or compounds are selected from DNA molecules,RNA molecules, peptide nucleic acids, peptides, proteins, mimetics,agonists, antagonists, antibodies, immunoglobulins, inhibitors, anddrugs. In another aspect, the ligand is used to treat a subject withcell proliferative disorders, particularly cancers of the colon, breast,ovary, uterus, prostate, adrenal gland, and thyroid, and thyroidfollicular adenoma and thyroid lymphocytic thyroiditis.

[0017] The invention provides a method of using a mammalian protein toscreen a subject sample for antibodies which specifically bind theprotein comprising isolating antibodies from the subject sample,contacting the isolated antibodies with the protein under conditionsthat allow specific binding, dissociating the antibody from thebound-protein, and comparing the quantity of antibody with knownstandards, wherein the presence or quantity of antibody is diagnostic ofcell proliferative disorders, particularly cancers of the colon, breast,ovary, uterus, prostate, adrenal gland, and thyroid, and thyroidfollicular adenoma and thyroid lymphocytic thyroiditis.

[0018] The invention also provides a method of using a mammalian proteinto prepare and purify antibodies comprising immunizing a animal with theprotein under conditions to elicit an antibody response, isolatinganimal antibodies, attaching the protein to a substrate, contacting thesubstrate with isolated antibodies under conditions to allow specificbinding to the protein, dissociating the antibodies from the protein,thereby obtaining purified antibodies.

[0019] The invention provides a purified antibody which bindsspecifically to a protein which is expressed in cell proliferative cellproliferative disorders, particularly cancers of the colon, breast,ovary, uterus, prostate, adrenal gland, and thyroid, and thyroidfollicular adenoma and thyroid lymphocytic thyroiditis. The inventionalso provides a method of using an antibody to diagnose cellproliferative disorders, particularly cancers of the colon, breast,ovary, uterus, prostate, adrenal gland, and thyroid, and thyroidfollicular adenoma and thyroid lymphocytic thyroiditis comprisingcombining the antibody comparing the quantity of bound antibody to knownstandards, thereby establishing the presence of cell proliferativedisorders, particularly cancers of the colon, breast, ovary, uterus,prostate, adrenal gland, and thyroid, and thyroid follicular adenoma andthyroid lymphocytic thyroiditis. The invention further provides a methodof using an antibody to treat cell proliferative disorders, particularlycancers of the colon, breast, ovary, uterus, prostate, adrenal gland,and thyroid, and thyroid follicular adenoma and thyroid lymphocyticthyroiditis comprising administering to a patient in need of suchtreatment a pharmaceutical composition comprising the purified antibody.

[0020] The invention provides a method for inserting a marker gene intothe genomic DNA of a mammal to disrupt the expression of the endogenouspolynucleotide. The invention also provides a method for using a cDNA toproduce a mammalian model system, the method comprising constructing avector containing the cDNA selected from SEQ ID NOs:2-49, transformingthe vector into an embryonic stem cell, selecting a transformedembryonic stem, microinjecting the transformed embryonic stem cell intoa mammalian blastocyst, thereby forming a chimeric blastocyst,transferring the chimeric blastocyst into a pseudopregnant dam, whereinthe dam gives birth to a chimeric offspring containing the cDNA in itsgerm line, and breeding the chimeric mammal to produce a homozygous,mammalian model system.

BRIEF DESCRIPTION OF THE FIGURES AND TABLE

[0021]FIGS. 1A, 1B, and 1C show the amino acid sequence (SEQ ID NO:1)and nucleic acid sequence (SEQ ID NO:3) of VMP1. The translation wasproduced using MACDNASIS PRO software (Hitachi Software Engineering,South San Francisco Calif.).

[0022]FIGS. 2A, 2B, 2C, 2D, 2E, 2F, and 2G show the amino acid sequence(SEQ ID NO:2) and nucleic acid sequence (SEQ ID NO:26) of VMP2. Thetranslation was produced using MACDNASIS PRO software (Hitachi SoftwareEngineering).

[0023]FIGS. 3A and 3B demonstrate the conserved chemical and structuralsimilarities among the sequences and domains of VMP1 (743725; SEQ IDNO:1), C. boidinii PMP20 (g170899; SEQ ID NO:52), and Synechocystismembrane protein s111621 (g1652858; SEQ ID NO:53). The alignment wasproduced using the MEGALIGN program of LASERGENE software (DNASTAR,Madison Wis.).

[0024]FIGS. 4A, 4B, 4C, and 4D demonstrate the conserved chemical andstructural similarities among the sequences and domains of VMP2(2822412; SEQ ID NO:2), human KIAA0255 (g1665777; SEQ ID NO:54), andyeast endosome EMP70 protein precursor (g2131246; SEQ ID NO:55),produced using the MEGALIGN program of LASERGENE software (DNASTAR).

[0025]FIGS. 5A and 5B demonstrate the conserved chemical and structuralsimilarities among the sequences and domains of human VMP2 (2822412; SEQID NO:2) and rat VMP2 (SEQ ID NO:51), produced using the MEGALIGNprogram of LASERGENE software (DNASTAR).

[0026] Tables 1 and 2 show the northern analysis for VMP1 produced usingthe LIFESEQ Gold database (Incyte Genomics, Palo Alto, Calif.). In Table1, the first column presents the tissue categories; the second column,the total number of clones in the tissue category; the third column, theratio of the number of libraries in which at least one transcript wasfound to the total number of libraries; the fourth column, absoluteclone abundance of the transcript; and the fifth column, percentabundance of the transcript. Table 2 shows expression of VMP1 in tissuefrom patients with cell proliferative disorders. The first column liststhe library name, the second column, the number of clones sequenced forthat library; the third column, the description of the tissue from whichthe library was derived; the fourth column, the absolute abundance ofthe transcript; and the fifth column, the percent abundance of thetranscript.

[0027] Tables 3 and 4 show the northern analysis for VMP2 produced usingthe LIFESEQ Gold database (Incyte Genomics, Palo Alto Calif.). In Table3, the first column presents the tissue categories; the second column,the total number of clones in the tissue category; the third column, theratio of the number of libraries in which at least one transcript wasfound to total number of libraries; the fourth column, the absoluteclone abundance of the transcript; and the fifth column, the percentabundance of the transcript. Table 4 shows expression of VMP2 in tissuefrom patients with cell proliferative disorders. The first column liststhe library name, the second column, the number of clones sequenced forthat library; the third column, description of the tissue from which thelibrary was derived; the fourth column, absolute clone abundance of thetranscript; and the fifth column, percent abundance of the transcript.

DESCRIPTION OF THE INVENTION

[0028] It is understood that this invention is not limited to theparticular machines, materials and methods described. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments and is not intended to limit the scopeof the present invention which will be limited only by the appendedclaims. As used herein, the singular forms “a”, “an”, and “the” includeplural reference unless the context clearly dictates otherwise. Forexample, a reference to “a host cell” includes a plurality of such hostcells known to those skilled in the art.

[0029] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. All publicationsmentioned herein are cited for the purpose of describing and disclosingthe cell lines, protocols, reagents and vectors which are reported inthe publications and which might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

[0030] Definitions

[0031] “VMP” refers to a substantially purified protein obtained fromany mammalian species, including bovine, canine, murine, ovine, porcine,rodent, simian, and preferably the human species, and from any source,whether natural, synthetic, semi-synthetic, or recombinant.

[0032] “Array” refers to an ordered arrangement of at least two cDNAs ona substrate. At least one of the cDNAs represents a control or standardsequence, and the other, a cDNA of diagnostic interest. The arrangementof from about two to about 40,000 cDNAs on the substrate assures thatthe size and signal intensity of each labeled hybridization complexformed between a cDNA and a sample nucleic acid is individuallydistinguishable.

[0033] The “complement” of a cDNA of the Sequence Listing refers to anucleic acid molecule which is completely complementary over its fulllength and which will hybridize to the cDNA or an mRNA under conditionsof high stringency.

[0034] “cDNA” refers to an isolated polynucleotide, nucleic acidmolecule, or any fragment or complement thereof. It may have originatedrecombinantly or synthetically, be double-stranded or single-stranded,represent coding and/or noncoding sequence, an exon with or without anintron from a genomic DNA molecule.

[0035] The phrase “cDNA encoding a protein” refers to a nucleic acidsequence that closely aligns with sequences which encode conservedregions, motifs or domains that were identified by employing analyseswell known in the art. These analyses include BLAST (Basic LocalAlignment Search Tool; Altschul (1993) J Mol Evol 36: 290-300; Altschulet al. (1990) J Mol Biol 215:403-410) which provides identity within theconserved region.

[0036] “Derivative” refers to a cDNA or a protein that has beensubjected to a chemical modification. Derivatization of a cDNA caninvolve substitution of a nontraditional base such as queosine or of ananalog such as hypoxanthine. These substitutions are well known in theart. Derivatization of a protein involves the replacement of a hydrogenby an acetyl, acyl, alkyl, amino, formyl, or morpholino group.Derivative molecules retain the biological activities of the naturallyoccurring molecules but may confer advantages such as longer lifespan orenhanced activity.

[0037] “Differential expression” refers to an increased, upregulated orpresent, or decreased, downregulated or absent, gene expression asdetected by the absence, presence, or at least two-fold changes in theamount of transcribed messenger RNA or translated protein in a sample.

[0038] “Disorder” refers to conditions, diseases or syndromes in whichthe cDNAs and VMP1 or VMP2 are differentially expressed such as cellproliferative disorders, particularly cancers of the colon, breast,ovary, uterus, prostate, adrenal gland, and thyroid including thyroidfollicular adenoma, thyroid lymphocytic thyroiditis, Crohn's disease,colon adenocarcinoma, breast papillomatosis, breast adenocarcinoma,ovary seroanaplastic carcinoma, ovary follicular cysts, cervixcervicitis, uterus serous papillary carcinoma, uterus endometrialadenocarcinoma, prostate adenofibromatous hyperplasia, and prostateadenocarcinoma.

[0039] “Fragment” refers to a chain of consecutive nucleotides fromabout 200 to about 700 base pairs in length. Fragments may be used inPCR or hybridization technologies to identify related nucleic acidmolecules and in binding assays to screen for a ligand. Nucleic acidsand their ligands identified in this manner are useful as therapeuticsto regulate replication, transcription or translation.

[0040] A “hybridization complex” is formed between a cDNA and a nucleicacid of a sample when the purines of one molecule hydrogen bond with thepyrimidines of the complementary molecule, e.g., 5′-A-G-T-C-3′ basepairs with 3′-T-C-A-G-5′. The degree of complementarity and the use ofnucleotide analogs affect the efficiency and stringency of hybridizationreactions.

[0041] “Ligand” refers to any agent, molecule, or compound which willbind specifically to a complementary site on a cDNA molecule orpolynucleotide, or to an epitope or a protein. Such ligands stabilize ormodulate the activity of polynucleotides or proteins and may be composedof inorganic or organic substances including nucleic acids, proteins,carbohydrates, fats, and lipids.

[0042] “Oligonucleotide” refers a single stranded molecule from about 18to about 60 nucleotides in length which may be used in hybridization oramplification technologies or in regulation of replication,transcription or translation. Substantially equivalent terms areamplimer, primer, and oligomer.

[0043] “Portion” refers to any part of a protein used for any purpose;but especially, to an epitope for the screening of ligands or for theproduction of antibodies.

[0044] “Post-translational modification” of a protein can involvelipidation, glycosylation, phosphorylation, acetylation, racemization,proteolytic cleavage, and the like. These processes may occursynthetically or biochemically. Biochemical modifications will vary bycellular location, cell type, pH, enzymatic milieu, and the like.

[0045] “Probe” refers to a cDNA that hybridizes to at least one nucleicacid in a sample. Where targets are single stranded, probes arecomplementary single strands. Probes can be labeled with reportermolecules for use in hybridization reactions including Southern,northern, in situ, dot blot, array, and like technologies or inscreening assays.

[0046] “Protein” refers to a polypeptide or any portion thereof. A“portion” of a protein refers to that length of amino acid sequencewhich would retain at least one biological activity, a domain identifiedby PFAM or PRINTS analysis or an antigenic epitope of the proteinidentified using Kyte-Doolittle algorithms of the PROTEAN program(DNASTAR). An “oligopeptide” is an amino acid sequence from about fiveresidues to about 15 residues that is used as part of a fusion proteinto produce an antibody.

[0047] “Purified” refers to any molecule or compound that is separatedfrom its natural environment and is from about 60% free to about 90%free from other components with which it is naturally associated.

[0048] “Sample” is used in its broadest sense as containing nucleicacids, proteins, antibodies, and the like. A sample may comprise abodily fluid; the soluble fraction of a cell preparation, or an aliquotof media in which cells were grown; a chromosome, an organelle, ormembrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA insolution or bound to a substrate; a cell; a tissue; a tissue print; afingerprint, buccal cells, skin, or hair; and the like.

[0049] “Specific binding” refers to a special and precise interactionbetween two molecules which is dependent upon their structure,particularly their molecular side groups. For example, the intercalationof a regulatory protein into the major groove of a DNA molecule, thehydrogen bonding along the backbone between two single stranded nucleicacids, or the binding between an epitope of a protein and an agonist,antagonist, or antibody.

[0050] “Similarity” as applied to sequences, refers to thequantification (usually percentage) of nucleotide or residue matchesbetween at least two sequences aligned using a standardized algorithmsuch as Smith-Waterman alignment (Smith and Waterman (1981) J Mol Biol147:195-197) or BLAST2 (Altschul et al. (1997) Nucleic Acids Res25:3389-3402). BLAST2 may be used in a standardized and reproducible wayto insert gaps in one of the sequences in order to optimize alignmentand to achieve a more meaningful comparison between them.

[0051] “Substrate” refers to any rigid or semi-rigid support to whichcDNAs or proteins are bound and includes membranes, filters, chips,slides, wafers, fibers, magnetic or nonmagnetic beads, gels, capillariesor other tubing, plates, polymers, and microparticles with a variety ofsurface forms including wells, trenches, pins, channels and pores.

[0052] “Variant” refers to molecules that are recognized variations of acDNA or a protein encoded by the cDNA. Splice variants may be determinedby BLAST score, wherein the score is at least 100, and most preferablyat least 400. Allelic variants have a high percent identity to the cDNAsand may differ by about three bases per hundred bases. “Singlenucleotide polymorphism” (SNP) refers to a change in a single base as aresult of a substitution, insertion or deletion. The change may beconservative (purine for purine) or non-conservative (purine topyrimidine) and may or may not result in a change in an encoded aminoacid or its secondary, tertiary, or quaternary structure.

[0053] The Invention

[0054] The invention is based on the discovery of cDNAs which encode VMPand on the use of the cDNAs, or fragments thereof, and proteins, orportions thereof, directly or as compositions in the characterization,diagnosis, and treatment of cell proliferative disorders.

[0055] Nucleic acids encoding the VMP1 of the present invention werefirst identified in Incyte Clone 743725 from the brain cDNA library(BRAITUT01) using a computer search for amino acid sequence alignments.A consensus sequence, SEQ ID NO:3, was derived from the followingoverlapping and/or extended nucleic acid sequences (SEQ ID NOs:4-10):Incyte Clones 743725H1 (BRAITUT01), 2521256H1 (BRAITUT21), 602137H1(BRSTNOT02), 2373064H1 (ADRENOT07), 1732084X15 (BRSTTUT08),911226H1(STOMNOT02), and 2226546H1 (SEMVNOT01). Table 1 shows expression of theVMP1 transcript across the tissue categories (also listed in ExampleVIII). VMP1 is expressed in various tissues including the cardiovascularsystem, connective tissue, digestive system, endocrine glands, exocrineglands, female and male reproductive tissues, hemic and immune system,nervous system, respiratory system and urinary tract. As shown in Table1, expression is low or absent in germ cell tumors, sense organs (eye,cochlea, and olfactory epithelia) and stomatognathic tumors. Table 2shows expression of the transcript in adrenal and thyroid tissues,particularly from patients with cell proliferative disorders. VMP1 showshigh expression in libraries from adrenal gland tissue (ADRENOT14,ADRENOT11, ADRENOT07, and ADRENOT09) and in libraries from patients withadrenal tumors (ADRETUT01 and ADRETUT07). VMP1 shows overexpression in alibrary from thyroid tissue from a patient with follicular adenoma(THYRTUT03). VMP1 shows overexpression in libraries from thyroid tissuefrom patients with lymphocytic thyroiditis (THYRNOT08 and THYRNOT10).

[0056] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A,1B, and 1C. VMP1 is 214 amino acids in length and has a predicted sizeof 22 kDa. VMP1 has two potential cyclic AMP- or cyclic GMP-dependentprotein kinase phosphorylation sites at residues S34 and S182; and twopotential casein kinase II phosphorylation sites at residues S34 andS127. PFAM analysis indicates that the region of VMP1 from V58 to N209is similar to an AhpC-TSA domain. The AhpC-TSA domain is found inantioxidant enzymes. As shown in FIGS. 3A and 3B, VMP1 has chemical andstructural homology with C. boidinii PMP20 (g170899; SEQ ID NO:52), andSynechocystis membrane protein s111621 (g1652858; SEQ ID NO:53). Inparticular, VMP1 and C. boidinii PMP20 share 29% identity over 172residues, have one potential casein kinase II phosphorylation site, andhave similar isoelectric points, 8.7 and 9.8, respectively. Usefulantigenic epitopes extend from A28 to A49, E66 to A79, G174 to S182, andL193 to L202; and a biologically active portion of VMP1 extends from V58to N209. An antibody which specifically binds VMP1 is useful in assaysto diagnose adrenal and thyroid disorders, particularly follicularadenoma and lymphocytic thyroiditis.

[0057] Nucleic acids encoding the VMP2 of the present invention werefirst identified in Incyte Clone 2822412 from the adrenalpheochromocytoma cDNA library (ADRETUT06) using a computer search foramino acid sequence alignments. A consensus sequence, SEQ ID NO:26, wasderived from the following overlapping and/or extended nucleic acidsequences (SEQ ID NOs:27-37): Incyte Clones 2822412H1 (ADRETIUT06),3236331H1 (COLNUCT03), 269777R1 (HNT2NOT01), 1359919F1(LUNGNOT12),770535R1 (COLNCRT01), 002505H1 (HMC1NOT01), 896216H1(BRSTNOT05), 741936H1 (PANCNOT04), 2112041H1 (BRAITUT03), 2132059R6(OVARNOT03), and 1609872X13 (COLNTUT06). Table 3 shows expression of thetranscript across the tissue categories. VMP2 is expressed in varioustissues including the digestive system, endocrine glands, exocrineglands, female and male reproductive tissues, respiratory system,stomatognathic system, and urinary tract. As shown in Table 3,expression is low or absent in germ cell tumors and sense organs (eye,cochlea, and olfactory epithelia). Table 4 shows expression of the VMP2transcript in tissue from patients with cell proliferative disorders.VMP2 shows overexpression in a library from colon tissue from a patientwith Crohn's disease (COLNCRT01) compared to a library from matched (m)microscopically normal tissue from the same donor (COLNNOT05). VMP2shows overexpression in a library from a patient with colon tumors(COLSTUT01). VMP2 shows overexpression in a library from breast tissuefrom a patient with fibrocystic breast disease (BRSTNOT12). VMP2 showsoverexpression in a library from breast tissue from a patient withadenocarcinoma (BRSTTUT02) compared to a library from matched (m)microscopically normal tissue from the same donor (BRSTNOT03). VMP2shows overexpression in a library from breast tissue from a patient withhigh vascular density cancer (BRSTTUT25), and in a library from breasttissue from a patient with papillomatosis (BRSTNOT16). VMP2 showsoverexpression in libraries from tissue from patients with ovariantumors (OVARTUT03, OVARTUT10, OVARTUP02, and OVARTUP06). VMP2 showsoverexpression in libraries from tissue from patients with uterinetumors (UTRSTUP05 and URTSTUP02). VMP2 shows overexpression in a libraryfrom cervex tissue from a patient with cervicitis (CERVNOT 01). VMP2shows overexpression in libraries from tissue from patients withprostate tumors (PROSTUP04, PROSTUT13, PROSTUT18, PROSTUT10, PROSTUT04,and PROSTUT01) compared to libraries from nontumorous tissues (PROSNOT02and PROSNOT19). VMP2 shows overexpression in libraries from tissue frompatients with prostate adenofibromatous hyperplasia (PROSTMC01,PROSDIN01, PROSTNOT16, PROSNOT18, PROSTMC02, PROSTMY01, PROSNOT06, andPROSNOT15).

[0058] In another embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:2, as shown in FIGS. 2A,2B, 2C, 2D, 2E, 2F, and 2G. VMP2 is 663 amino acids in length and has apredicted size of 76 kDa. VMP2 has two potential cyclic AMP- or cyclicGMP-dependent protein kinase phosphorylation sites at residues S93 andT119; six potential casein kinase II phosphorylation sites at residuesT79, T243, S274, S285, S338, and T568; six potential protein kinase Cphosphorylation sites at residues S2, T119, T130, T185, S239, and S258;one potential tyrosine kinase phosphorylation site at residue Y517; andten potential hydrophobic transmembrane domains between residues R15 andV27, W301 and R324, L364 and N395, L399 and F421, L435 and S462, S462and Y490, L521 and 1546, M554 and L581, L594 and T618, and T618 andD663. As shown in FIGS. 4A, 4B, 4C, and 4D, VMP2 has chemical andstructural homology with human KIAA0255 (g1665777; SEQ ID NO:54) andyeast endosome EMP70 protein precursor (g2131246; SEQ ID NO:55). Inparticular, VMP2 and human KIAA0255 share 41% identity over 663residues, one potential casein kinase II phosphorylation site, twopotential protein kinase C phosphorylation sites, and one potentialtyrosine kinase phosphorylation site. In addition, VMP2 and humanKIAA0255 have similar potential isoelectric points, 7.1 and 6.2,respectively. Useful antigenic epitopes extend from P237 to K266 andfrom G492 to G523. An antibody which specifically binds VMP2 is usefulin assays to diagnose cell proliferative disorders, particularly cancersof the colon, breast ovary, uterus, and prostate.

[0059] Mammalian variants of the cDNAs encoding VMP1 or VMP2 wereidentified using BLAST2 with default parameters and the ZOOSEQ databases(Incyte Genomics). These preferred variants have from about 80% to about100% identity as shown in the table below. The first column shows theSEQ ID for the human cDNA (SEQ ID_(H)); the second column, the SEQ IDfor the variant cDNAs (SEQ IDvar); the third column, the clone numberfor the variant cDNAs; the fourth column, the library name; the fifthcolumn, the alignment of the variant cDNA to the human cDNA; and thesixth column, the percent identity to the human cDNA. cDNA Librarieswere isolated from monkey, mouse, rat, and dog tissues. Library SEQID_(H) SEQ ID_(var) Clone_(var) Name Nt_(H) Alignment Identity 3 11700459782H1 MNBFNOT01 194-447 95% 3 12 700711869H1 MNBFNOT02 113-378 91%3 13 700711005H2 MNBFNOT02 104-345 90% 3 14 700712820H1 MNBFNOT02 83-324 89% 3 15 700718769H1 MNBCNOT01  72-321 88% 3 16 700715209H1MNBCNOT01 617-759 93% 3 17 700708715H1 MNBFNOT01 194-312 94% 3 18701738592T1 MNBCNON01  440-549,  96%, 626-705 92% 3 19 700459993H2MNBFNOT02  49-259 85% 3 20 701087440H1 MOLUDIT05 264-439 88% 3 21701253435H1 MOLUDIT07 264-411 87% 3 22 701424530H1 MOAPUNT01 264-372 89%3 23 701423726H1 MOAPUNT01 264-321 91% 3 24 702154193H1 RABRTXT11344-761 84% 3 25 702242583H1 RAOVNOT01 275-530 85% 26 38 700708848H1MNBFNOT01 1375-1460 95% 26 39 700108409H2 MOOSUN7RO1 1473-1761 93% 26 40701091450H1 MOLUDITO5  857-1144 90% 26 41 701080359H1 MOLUDITO32225-2480 88% 26 42 702775324H1 CNLINOT06 1808-2243 96% 26 43702237054H1 RALUNOT02  695-1234 99% 26 44 702212516H1 RALUNOT01 543-1066 99% 26 45 702599525T1 RAKINOT02 1172-1716 97% 26 46700228705H1 RACONOT01  1-293 100%  26 47 700230086H1 RACONOT01 340-62699% 26 48 700776642H1 RAPINOT02 270-536 100%  26 49 700545190H1RASPNOT01 1684-1980 98% 26 50 700228705.con   1-2805 80%

[0060] These cDNAs are particularly useful for producing transgenic celllines or organisms which model human disorders and upon which potentialtherapeutic treatments for such disorders may be tested.

[0061] Nucleic acids encoding the rat variant of VMP2, 700228705.con, ofthe present invention, were first identified in Incyte Clone 700228705from the rat colon cDNA library (RACONOT01) using a computer search fornucleic acid sequence alignments. A consensus sequence, SEQ ID NO:50,was derived from the following overlapping and/or extended nucleic acidsequences: Incyte Clones 702237054H1 (RALUNOT02), 702212516H1(RALUNOT01), 702599525T1 (RAKINOT02), 700228705H1 (RACONOT01),700230086H1 (RACONOT01), 700776642H1 (RAPINOT02), and 700545190H1(RASPNOT01).

[0062] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:51, as shown in FIGS. 5Aand 5B. The rat variant of VMP2 is 394 amino acids in length and shares98% identity with human VMP2.

[0063] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of cDNAsencoding VMP, some bearing minimal similarity to the cDNAs of any knownand naturally occurring gene, may be produced. Thus, the inventioncontemplates each and every possible variation of cDNA that could bemade by selecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to the polynucleotide encoding naturally occurring VMP,and all such variations are to be considered as being specificallydisclosed.

[0064] The cDNAs and fragments thereof (SEQ ID NOs:3-50) may be used inhybridization, amplification, and screening technologies to identify anddistinguish among SEQ ID NO:2 and related molecules in a sample. Themammalian cDNAs may be used to produce transgenic cell lines ororganisms which are model systems for human cell proliferativedisorders, particularly cancers of the colon, breast, ovary, uterus,prostate, adrenal gland, and thyroid and upon which the toxicity andefficacy of potential therapeutic treatments may be tested. Toxicologystudies, clinical trials, and subject/patient treatment profiles may beperformed and monitored using the cDNAs, proteins, antibodies andmolecules and compounds identified using the cDNAs and proteins of thepresent invention.

[0065] Characterization and Use of the Invention

[0066] cDNA Libraries

[0067] In a particular embodiment disclosed herein, mRNA was isolatedfrom mammalian cells and tissues using methods which are well known tothose skilled in the art and used to prepare the cDNA libraries. TheIncyte clones listed above were isolated from mammalian cDNA libraries.Three library preparations representative of the invention are describedin the EXAMPLES below. The consensus sequences were chemically and/orelectronically assembled from fragments including Incyte clones andextension and/or shotgun sequences using computer programs such as PHRAP(P Green, University of Washington, Seattle Wash.), and AUTOASSEMBLERapplication (Applied Biosystems, Foster City Calif.). Clones, extensionand/or shotgun sequences are electronically assembled into clustersand/or master clusters.

[0068] Sequencing

[0069] Methods for sequencing nucleic acids are well known in the artand may be used to practice any of the embodiments of the invention.These methods employ enzymes such as the Klenow fragment of DNApolymerase I, SEQUENASE, Taq DNA polymerase and thermostable T7 DNApolymerase (Amersham Pharmacia Biotech (APB), Piscataway N.J.), orcombinations of polymerases and proofreading exonucleases such as thosefound in the ELONGASE amplification system (Life Technologies,Gaithersburg Md.). Preferably, sequence preparation is automated withmachines such as the MICROLAB 2200 system (Hamilton, Reno Nev.) and theDNA ENGINE thermal cycler (MJ Research, Watertown Mass.). Machinescommonly used for sequencing include the ABI PRISM 3700, 377 or 373 DNAsequencing systems (Applied Biosystems), the MEGABACE 1000 DNAsequencing system (APB), and the like. The sequences may be analyzedusing a variety of algorithms well known in the art and described inAusubel et al (1997; Short Protocols in Molecular Biology, John Wiley &Sons, New York N.Y., unit 7.7) and in Meyers (1995; Molecular Biologyand Biotechnology, Wiley VCH, New York, N.Y. pp. 856-853).

[0070] Shotgun sequencing may also be used to complete the sequence of aparticular cloned insert of interest. Shotgun strategy involves randomlybreaking the original insert into segments of various sizes and cloningthese fragments into vectors. The fragments are sequenced andreassembled using overlapping ends until the entire sequence of theoriginal insert is known. Shotgun sequencing methods are well known inthe art and use thermostable DNA polymerases, heat-labile DNApolymerases, and primers chosen from representative regions flanking thecDNAs of interest. Incomplete assembled sequences are inspected foridentity using various algorithms or programs such as CONSED (Gordon(1998) Genome Res 8:195-202) which are well known in the art.Contaminating sequences including vector or chimeric sequences ordeleted sequences can be removed or restored, respectively, organizingthe incomplete assembled sequences into finished sequences.

[0071] Extension of a Nucleic Acid Sequence

[0072] The sequences of the invention may be extended using variousPCR-based methods known in the art. For example, the XL-PCR kit (AppliedBiosystems), nested primers, and commercially available cDNA or genomicDNA libraries may be used to extend the nucleic acid sequence. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO primer analysis software (Molecular BiologyInsights, Cascade Colo.) to be about 22 to 30 nucleotides in length, tohave a GC content of about 50% or more, and to anneal to a targetmolecule at temperatures from about 55 C. to about 68 C. When extendinga sequence to recover regulatory elements, it is preferable to usegenomic, rather than cDNA libraries.

[0073] Hybridization

[0074] The cDNA and fragments thereof can be used in hybridizationtechnologies for various purposes. A probe may be designed or derivedfrom unique regions such as the 5′ regulatory region or from anonconserved region (i.e., 5′ or 3′ of the nucleotides encoding theconserved catalytic domain of the protein) and used in protocols toidentify naturally occurring molecules encoding the VMP1 or VMP2,allelic variants, or related molecules. The probe may be DNA or RNA, maybe single stranded and should have at least 50% sequence identity to anyof the nucleic acid sequences, SEQ ID NOs:2-50. Hybridization probes maybe produced using oligolabeling, nick translation, end-labeling, or PCRamplification in the presence of a reporter molecule. A vectorcontaining the cDNA or a fragment thereof may be used to produce an mRNAprobe in vitro by addition of an RNA polymerase and labeled nucleotides.These procedures may be conducted using commercially available kits suchas those provided by APB.

[0075] The stringency of hybridization is determined by G+C content ofthe probe, salt concentration, and temperature. In particular,stringency can be increased by reducing the concentration of salt orraising the hybridization temperature. In solutions used for somemembrane based hybridizations, addition of an organic solvent such asformamide allows the reaction to occur at a lower temperature.Hybridization can be performed at low stringency with buffers, such as5×SSC with 1% sodium dodecyl sulfate (SDS) at 60 C., which permits theformation of a hybridization complex between nucleic acid sequences thatcontain some mismatches. Subsequent washes are performed at higherstringency with buffers such as 0.2×SSC with 0.1% SDS at either 45 C.(medium stringency) or 68 C. (high stringency). At high stringency,hybridization complexes will remain stable only where the nucleic acidsare completely complementary. In some membrane-based hybridizations,preferably 35% or most preferably 50%, formamide can be added to thehybridization solution to reduce the temperature at which hybridizationis performed, and background signals can be reduced by the use of otherdetergents such as Sarkosyl or TRITON X-100 (Sigma-Aldrich, St. LouisMo.) and a blocking agent such as denatured salmon sperm DNA. Selectionof components and conditions for hybridization are well known to thoseskilled in the art and are reviewed in Ausubel (supra) and Sambrook etal. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview N.Y.

[0076] Arrays may be prepared and analyzed using methods known in theart. Oligonucleotides may be used as either probes or targets in anarray. The array can be used to monitor the expression level of largenumbers of genes simultaneously and to identify genetic variants,mutations, and single nucleotide polymorphisms. Such information may beused to determine gene function; to understand the genetic basis of acondition, disease, or disorder; to diagnose a condition, disease, ordisorder; and to develop and monitor the activities of therapeuticagents. (See, e.g., Brennan et al. (1995) U.S. Pat. No. 5,474,796;Schena et al. (1996) Proc Natl Acad Sci 93:10614-10619; Baldeschweileret al. (1995) PCT application WO95/251116; Shalon et al. (1995) PCTapplication WO95/35505; Heller et al. (1997) Proc Natl Acad Sci94:2150-2155; and Heller et al. (1997) U.S. Pat. No. 5,605,662.)

[0077] Hybridization probes are also useful in mapping the naturallyoccurring genomic sequence. The probes may be hybridized to: 1) aparticular chromosome, 2) a specific region of a chromosome, or 3) anartificial chromosome construction such as human artificial chromosome(HAC), yeast artificial chromosome (YAC), bacterial artificialchromosome (BAC), bacterial P1 construction, or single chromosome cDNAlibraries.

[0078] Expression

[0079] Any one of a multitude of cDNAs encoding VMP may be cloned into avector and used to express the protein, or portions thereof, in hostcells. The nucleic acid sequence can be engineered by such methods asDNA shuffling (U.S. Pat. No. 5,830,721) and site-directed mutagenesis tocreate new restriction sites, alter glycosylation patterns, change codonpreference to increase expression in a particular host, produce splicevariants, extend half-life, and the like. The expression vector maycontain transcriptional and translational control elements (promoters,enhancers, specific initiation signals, and polyadenylated 3′ sequence)from various sources which have been selected for their efficiency in aparticular host. The vector, cDNA, and regulatory elements are combinedusing in vitro recombinant DNA techniques, synthetic techniques, and/orin vivo genetic recombination techniques well known in the art anddescribed in Sambrook (supra, ch. 4, 8, 16 and 17).

[0080] A variety of host systems may be transformed with an expressionvector. These include, but are not limited to, bacteria transformed withrecombinant bacteriophage, plasmid, or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemstransformed with baculovirus expression vectors; plant cell systemstransformed with expression vectors containing viral and/or bacterialelements, or animal cell systems (Ausubel supra, unit 16). For example,an adenovirus transcription/translation complex may be utilized inmammalian cells. After sequences are ligated into the E1 or E3 region ofthe viral genome, the infective virus is used to transform and expressthe protein in host cells. The Rous sarcoma virus enhancer or SV40 orEBV-based vectors may also be used for high-level protein expression.

[0081] Routine cloning, subcloning, and propagation of nucleic acidsequences can be achieved using the multifunctional PBLUESCRIPT vector(Stratagene, La Jolla Calif.) or PSPORT1 plasmid (Life Technologies).Introduction of a nucleic acid sequence into the multiple cloning siteof these vectors disrupts the lacZ gene and allows colorimetricscreening for transformed bacteria. In addition, these vectors may beuseful for in vitro transcription, dideoxy sequencing, single strandrescue with helper phage, and creation of nested deletions in the clonedsequence.

[0082] For long term production of recombinant proteins, the vector canbe stably transformed into cell lines along with a selectable or visiblemarker gene on the same or on a separate vector. After transformation,cells are allowed to grow for about 1 to 2 days in enriched media andthen are transferred to selective media. Selectable markers,antimetabolite, antibiotic, or herbicide resistance genes, conferresistance to the relevant selective agent and allow growth and recoveryof cells which successfully express the introduced sequences. Resistantclones identified either by survival on selective media or by theexpression of visible markers, such as anthocyanins, green fluorescentprotein (GFP), βglucuronidase, luciferase and the like, may bepropagated using culture techniques. Visible markers are also used toquantify the amount of protein expressed by the introduced genes.Verification that the host cell contains the desired mammalian cDNA isbased on DNA-DNA or DNA-RNA hybridizations or PCR amplificationtechniques.

[0083] The host cell may be chosen for its ability to modify arecombinant protein in a desired fashion. Such modifications includeacetylation, carboxylation, glycosylation, phosphorylation, lipidation,acylation and the like. Post-translational processing which cleaves a“prepro” form may also be used to specify protein targeting, folding,and/or activity. Different host cells available from the ATCC (ManassasVa.) which have specific cellular machinery and characteristicmechanisms for post-translational activities may be chosen to ensure thecorrect modification and processing of the recombinant protein.

[0084] Recovery of Proteins from Cell Culture

[0085] Heterologous moieties engineered into a vector for ease ofpurification include glutathione S-transferase (GST), 6xHis, FLAG, MYC,and the like. GST and 6-His are purified using commercially availableaffinity matrices such as immobilized glutathione and metal-chelateresins, respectively. FLAG and MYC are purified using commerciallyavailable monoclonal and polyclonal antibodies. For ease of separationfollowing purification, a sequence encoding a proteolytic cleavage sitemay be part of the vector located between the protein and theheterologous moiety. Methods for recombinant protein expression andpurification are discussed in Ausubel (supra, unit 16) and arecommercially available.

[0086] Chemical Synthesis of Peptides

[0087] Proteins or portions thereof may be produced not only byrecombinant methods, but also by using chemical methods well known inthe art. Solid phase peptide synthesis may be carried out in a batchwiseor continuous flow process which sequentially adds α-amino- and sidechain-protected amino acid residues to an insoluble polymeric supportvia a linker group. A linker group such as methylamine-derivatizedpolyethylene glycol is attached to poly(styrene-co-divinylbenzene) toform the support resin. The amino acid residues are N-α-protected byacid labile Boc (t-butyloxycarbonyl) or base-labile Fmoc(9-fluorenylmethoxycarbonyl). The carboxyl group of the protected aminoacid is coupled to the amine of the linker group to anchor the residueto the solid phase support resin. Trifluoroacetic acid or piperidine areused to remove the protecting group in the case of Boc or Fmoc,respectively. Each additional amino acid is added to the anchoredresidue using a coupling agent or pre-activated amino acid derivative,and the resin is washed. The full length peptide is synthesized bysequential deprotection, coupling of derivitized amino acids, andwashing with dichloromethane and/or N,N-dimethylformamide. The peptideis cleaved between the peptide carboxy terminus and the linker group toyield a peptide acid or amide. (Novabiochem 1997/98 Catalog and PeptideSynthesis Handbook, San Diego Calif. pp. S1-S20). Automated synthesismay also be carried out on machines such as the ABI 431A peptidesynthesizer (Applied Biosystems). A protein or portion thereof may besubstantially purified by preparative high performance liquidchromatography and its composition confirmed by amino acid analysis orby sequencing (Creighton (1984) Proteins, Structures and MolecularProperties, W H Freeman, New York N.Y.).

[0088] Preparation and Screening of Antibodies

[0089] Various hosts including goats, rabbits, rats, mice, humans, andothers may be immunized by injection with VMP or any portion thereof.Adjuvants such as Freund's, mineral gels, and surface active substancessuch as lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, keyhole limpet hemacyanin (KLH), and dinitrophenol may beused to increase immunological response. The oligopeptide, peptide, orportion of protein used to induce antibodies should consist of at leastabout five amino acids, more preferably ten amino acids, which areidentical to a portion of the natural protein. Oligopeptides may befused with proteins such as KLH in order to produce antibodies to thechimeric molecule.

[0090] Monoclonal antibodies may be prepared using any technique whichprovides for the production of antibodies by continuous cell lines inculture. These include, but are not limited to, the hybridoma technique,the human B-cell hybridoma technique, and the EBV-hybridoma technique.(See, e.g., Kohler et al. (1975) Nature 256:495-497; Kozbor et al.(1985) J. Immunol Methods 81:31-42; Cote et al. (1983) Proc Natl AcadSci 80:2026-2030; and Cole et al. (1984) Mol Cell Biol 62:109-120.)

[0091] Alternatively, techniques described for the production of singlechain antibodies may be adapted, using methods known in the art, toproduce epitope specific single chain antibodies. Antibody fragmentswhich contain specific binding sites for epitopes of the protein mayalso be generated. For example, such fragments include, but are notlimited to, F(ab′)2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huseet al. (1989) Science 246:1275-1281.)

[0092] VMP or a portion thereof may be used in screening assays ofphagemid or B-lymphocyte immunoglobulin libraries to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoassays using either polyclonal or monoclonal antibodieswith established specificities are well known in the art. Suchimmunoassays typically involve the measurement of complex formationbetween the protein and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes is preferred, but a competitive bindingassay may also be employed (Pound (1998) Immunochemical Protocols,Humana Press, Totowa N.J.).

[0093] Labeling of Molecules for Assay

[0094] A wide variety of reporter molecules and conjugation techniquesare known by those skilled in the art and may be used in various nucleicacid, amino acid, and antibody assays. Synthesis of labeled moleculesmay be achieved using commercially available kits (Promega, MadisonWis.) for incorporation of a labeled nucleotide such as ³²P-dCTP (APB),Cy3-dCTP or Cy5-dCTP (Operon Technologies, Alameda Calif.), or aminoacid such as ³⁵S-methionine (APB). Nucleotides and amino acids may bedirectly labeled with a variety of substances including fluorescent,chemiluminescent, or chromogenic agents, and the like, by chemicalconjugation to amines, thiols and other groups present in the moleculesusing reagents such as BIODIPY or FITC (Molecular Probes, Eugene Oreg.).

[0095] Diagnostics

[0096] The cDNAs, fragments, oligonucleotides, complementary RNA and DNAmolecules, and PNAs and may be used to detect and quantify differentialgene expression, absence/presence vs. excess, expression of mRNAs or tomonitor mRNA levels during therapeutic intervention. Similarlyantibodies which specifically bind VMP may be used to quantitate theprotein. Disorders associated with differential expression include cellproliferative disorders, particularly cancers of the colon, breast,ovary, uterus, prostate, adrenal gland, and thyroid. The diagnosticassay may use hybridization or amplification technology to compare geneexpression in a biological sample from a patient to standard samples inorder to detect differential gene expression. Qualitative orquantitative methods for this comparison are well known in the art.

[0097] For example, the cDNA or probe may be labeled by standard methodsand added to a biological sample from a patient under conditions for theformation of hybridization complexes. After an incubation period, thesample is washed and the amount of label (or signal) associated withhybridization complexes, is quantified and compared with a standardvalue. If complex formation in the patient sample is significantlyaltered (higher or lower) in comparison to either a normal or diseasestandard, then differential expression indicates the presence of adisorder.

[0098] In order to provide standards for establishing differentialexpression, normal and disease expression profiles are established. Thisis accomplished by combining a sample taken from normal subjects, eitheranimal or human, with a cDNA under conditions for hybridization tooccur. Standard hybridization complexes may be quantified by comparingthe values obtained using normal subjects with values from an experimentin which a known amount of a substantially purified sequence is used.Standard values obtained in this manner may be compared with valuesobtained from samples from patients who were diagnosed with a particularcondition, disease, or disorder. Deviation from standard values towardthose associated with a particular disorder is used to diagnose thatdisorder.

[0099] Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies and inclinical trial or to monitor the treatment of an individual patient.Once the presence of a condition is established and a treatment protocolis initiated, diagnostic assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in a normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0100] Immunological Methods

[0101] Detection and quantification of a protein using either specificpolyclonal or monoclonal antibodies are known in the art. Examples ofsuch techniques include enzyme-linked immunosorbent assays (ELISAs),radioimmunoassays (RIAs), and fluorescence activated cell sorting(FACS). A two-site, monoclonal-based immunoassay utilizing monoclonalantibodies reactive to two non-interfering epitopes is preferred, but acompetitive binding assay may be employed. (See, e.g., Coligan et al.(1997) Current Protocols in Immunology, Wiley-Interscience, New York,N.Y.; and Pound, supra.)

[0102] Therapeutics

[0103] Chemical and structural similarity exists between regions of VMP1(SEQ ID NO:1), C. boidinii PMP20 (g170899; SEQ ID NO:52), andSynechocystis membrane protein s 111621 (g1652858; SEQ ID NO:53) asshown in FIGS. 3A and 3B. Differential expression of VMP1 is associatedwith adrenal and thyroid disorders as shown in Tables 1 and 2. VMP1clearly plays a role in thyroid tumors and lymphocytic thyroiditis.

[0104] Chemical and structural similarity exists between regions of VMP2(SEQ ID NO:2), human KIAA0255 (g1665777; SEQ ID NO:54) and yeastendosome EMP70 protein precursor (g2131246; SEQ ID NO:55) as shown inFIGS. 4A, 4B, 4C, and 4D. Differential expression of VMP2 is associatedwith cell proliferative disorders as shown in Tables 3 and 4. VMP2clearly plays a role in cell proliferative disorders, particularlycancers of the colon, breast ovary, uterus, and prostate.

[0105] In the treatment of conditions associated with increasedexpression of VMP, it is desirable to decrease expression or proteinactivity. In one embodiment, the an inhibitor, antagonist or antibody ofthe protein may be administered to a subject to treat a conditionassociated with increased expression or activity. In another embodiment,a pharmaceutical composition comprising an inhibitor, antagonist orantibody in conjunction with a pharmaceutical carrier may beadministered to a subject to treat a condition associated with theincreased expression or activity of the endogenous protein. In anadditional embodiment, a vector expressing the complement of the cDNA orfragments thereof may be administered to a subject to treat thedisorder.

[0106] In the treatment of conditions associated with decreasedexpression of the protein; it is desirable to increase expression orprotein activity. In one embodiment, the protein, an agonist or enhancermay be administered to a subject to treat a condition associated withdecreased expression or activity. In another embodiment, apharmaceutical composition comprising the protein, an agonist orenhancer in conjunction with a pharmaceutical carrier may beadministered to a subject to treat a condition associated with thedecreased expression or activity of the endogenous protein. In anadditional embodiment, a vector expressing cDNA may be administered to asubject to treat the disorder.

[0107] Any of the cDNAs, complementary molecules, or fragments thereof,proteins or portions thereof, vectors delivering these nucleic acidmolecules or expressing the proteins, and their ligands may beadministered in combination with other therapeutic agents. Selection ofthe agents for use in combination therapy may be made by one of ordinaryskill in the art according to conventional pharmaceutical principles. Acombination of therapeutic agents may act synergistically to affecttreatment of a particular disorder at a lower dosage of each agent.

[0108] Modification of Gene Expression Using Nucleic Acids

[0109] Gene expression may be modified by designing complementary orantisense molecules (DNA, RNA, or PNA) to the control, 5′, 3′, or otherregulatory regions of the gene encoding VMP. Oligonucleotides designedwith reference to the transcription initiation site are preferred.Similarly, inhibition can be achieved using triple helix base-pairingwhich inhibits the binding of polymerases, transcription factors, orregulatory molecules (Gee et al. In: Huber and Carr (1994) Molecular andImmunologic Approaches, Futura Publishing, Mt. Kisco N.Y., pp. 163-177).A complementary molecule may also be designed to block translation bypreventing binding between ribosomes and mRNA. In one alternative, alibrary or plurality of cDNAs or fragments thereof may be screened toidentify those which specifically bind a regulatory, nontranslatedsequence.

[0110] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA followed by endonucleolytic cleavage at sitessuch as GUA, GUU, and GUC. Once such sites are identified, anoligonucleotide with the same sequence may be evaluated for secondarystructural features which would render the oligonucleotide inoperable.The suitability of candidate targets may also be evaluated by testingtheir hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0111] Complementary nucleic acids and ribozymes of the invention may beprepared via recombinant expression, in vitro or in vivo, or using solidphase phosphoramidite chemical synthesis. In addition, RNA molecules maybe modified to increase intracellular stability and half-life byaddition of flanking sequences at the 5′ and/or 3′ ends of the moleculeor by the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule.Modification is inherent in the production of PNAs and can be extendedto other nucleic acid molecules. Either the inclusion of nontraditionalbases such as inosine, queosine, and wybutosine, and or the modificationof adenine, cytidine, guanine, thymine, and uridine with acetyl-,methyl-, thio- groups renders the molecule less available to endogenousendonucleases.

[0112] Screening and Purification Assays

[0113] The cDNA encoding VMP may be used to screen a library ofmolecules or compounds for specific binding affinity. The libraries maybe aptamers, DNA molecules, RNA molecules, PNAs, peptides, proteins suchas transcription factors, enhancers, repressors, and other ligands whichregulate the activity, replication, transcription, or translation of thecDNA in the biological system. The assay involves combining the cDNA ora fragment thereof with the library of molecules under conditionsallowing specific binding, and detecting specific binding to identify atleast one molecule which specifically binds the single stranded or, ifappropriate, double stranded molecule.

[0114] In one embodiment, the cDNA of the invention may be incubatedwith a plurality of purified molecules or compounds and binding activitydetermined by methods well known in the art, e.g., a gel-retardationassay (U.S. Pat. No. 6,010,849) or a reticulocyte lysate transcriptionalassay. In another embodiment, the cDNA may be incubated with nuclearextracts from biopsied and/or cultured cells and tissues. Specificbinding between the cDNA and a molecule or compound in the nuclearextract is initially determined by gel shift assay and may be laterconfirmed by recovering and raising antibodies against that molecule orcompound. When these antibodies are added into the assay, they cause asupershift in the gel-retardation assay.

[0115] In another embodiment, the cDNA may be used to purify a moleculeor compound using affinity chromatography methods well known in the art.In one embodiment, the cDNA is chemically reacted with cyanogen bromidegroups on a polymeric resin or gel. Then a sample is passed over andreacts with or binds to the cDNA. The molecule or compound which isbound to the cDNA may be released from the cDNA by increasing the saltconcentration of the flow-through medium and collected.

[0116] In a further embodiment,the protein or a portion thereof may beused to purify a ligand from a sample. A method for using a mammalianprotein or a portion thereof to purify a ligand would involve combiningthe protein or a portion thereof with a sample under conditions to allowspecific binding, detecting specific binding between the protein andligand, recovering the bound protein, and using an appropriatechaotropic agent to separate the protein from the purified ligand.

[0117] In a preferred embodiment, VMP or a portion thereof may be usedto screen a plurality of molecules or compounds in any of a variety ofscreening assays. The portion of the protein employed in such screeningmay be free in solution, affixed to an abiotic or biotic substrate (e.g.borne on a cell surface), or located intracellularly. For example, inone method, viable or fixed prokaryotic host cells that are stablytransformed with recombinant nucleic acids that have expressed andpositioned a peptide on their cell surface can be used in screeningassays. The cells are screened against a plurality or libraries ofligands and the specificity of binding or formation of complexes betweenthe expressed protein and the ligand may be measured. Specific bindingbetween the protein and molecule may be measured. Depending on the kindof library being screened, the assay may be used to identify DNAmolecules, RNA molecules, peptide nucleic acids, peptides, proteins,mimetics, agonists, antagonists, antibodies, immunoglobulins,inhibitors, and drugs or any other ligand, which specifically binds theprotein.

[0118] In one aspect, this invention comtemplates a method for highthroughput screening using very small assay volumes and very smallamounts of test compound as described in U.S. Pat. No. 5,876,946,incorporated herein by reference. This method is used to screen largenumbers of molecules and compounds via specific binding. In anotheraspect, this invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable of binding theprotein specifically compete with a test compound capable of binding tothe protein or oligopeptide or portion thereof. Molecules or compoundsidentified by screening may be used in a mammalian model system toevaluate their toxicity, diagnostic, or therapeutic potential.

[0119] Pharmacology

[0120] Pharmaceutical compositions are those substances wherein theactive ingredients are contained in an effective amount to achieve adesired and intended purpose. The determination of an effective dose iswell within the capability of those skilled in the art. For anycompound, the therapeutically effective dose may be estimated initiallyeither in cell culture assays or in animal models. The animal model isalso used to achieve a desirable concentration range and route ofadministration. Such information may then be used to determine usefuldoses and routes for administration in humans.

[0121] A therapeutically effective dose refers to that amount of proteinor inhibitor which ameliorates the symptoms or condition. Therapeuticefficacy and toxicity of such agents may be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., ED₅₀ (the dose therapeutically effective in 50% of the population)and LD₅₀ (the dose lethal to 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index, and itmay be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceutical compositionswhich exhibit large therapeutic indexes are preferred. The data obtainedfrom cell culture assays and animal studies are used in formulating arange of dosage for human use.

[0122] Model Systems

[0123] Animal models may be used as bioassays where they exhibit aphenotypic response similar to that of humans and where exposureconditions are relevant to human exposures. Mammals are the most commonmodels, and most infectious agent, cancer, drug, and toxicity studiesare performed on rodents such as rats or mice because of low cost,availability, lifespan, reproductive potential, and abundant referenceliterature. Inbred and outbred rodent strains provide a convenient modelfor investigation of the physiological consequences of under- orover-expression of genes of interest and for the development of methodsfor diagnosis and treatment of diseases. A mammal inbred to over-expressa particular gene (for example, secreted in milk) may also serve as aconvenient source of the protein expressed by that gene.

[0124] Toxicology

[0125] Toxicology is the study of the effects of agents on livingsystems. The majority of toxicity studies are performed on rats or mice.Observation of qualitative and quantitative changes in physiology,behavior, homeostatic processes, and lethality in the rats or mice areused to generate a toxicity profile and to assess potential consequenceson human health following exposure to the agent.

[0126] Genetic toxicology identifies and analyzes the effect of an agenton the rate of endogenous, spontaneous, and induced genetic mutations.Genotoxic agents usually have common chemical or physical propertiesthat facilitate interaction with nucleic acids and are most harmful whenchromosomal aberrations are transmitted to progeny. Toxicologicalstudies may identify agents that increase the frequency of structural orfunctional abnormalities in the tissues of the progeny if administeredto either parent before conception, to the mother during pregnancy, orto the developing organism. Mice and rats are most frequently used inthese tests because their short reproductive cycle allows the productionof the numbers of organisms needed to satisfy statistical requirements.

[0127] Acute toxicity tests are based on a single administration of anagent to the subject to determine the symptomology or lethality of theagent. Three experiments are conducted: 1) an initial dose-range-findingexperiment, 2) an experiment to narrow the range of effective doses, and3) a final experiment for establishing the dose-response curve.

[0128] Subchronic toxicity tests are based on the repeatedadministration of an agent. Rat and dog are commonly used in thesestudies to provide data from species in different families. With theexception of carcinogenesis, there is considerable evidence that dailyadministration of an agent at high-dose concentrations for periods ofthree to four months will reveal most forms of toxicity in adultanimals.

[0129] Chronic toxicity tests, with a duration of a year or more, areused to demonstrate either the absence of toxicity or the carcinogenicpotential of an agent. When studies are conducted on rats, a minimum ofthree test groups plus one control group are used, and animals areexamined and monitored at the outset and at intervals throughout theexperiment.

[0130] Transgenic Animal Models

[0131] Transgenic rodents that over-express or under-express a gene ofinterest may be inbred and used to model human diseases or to testtherapeutic or toxic agents. (See, e.g., U.S. Pat. No. 5,175,383 andU.S. Pat. No. 5,767,337.) In some cases, the introduced gene may beactivated at a specific time in a specific tissue type during fetal orpostnatal development. Expression of the transgene is monitored byanalysis of phenotype, of tissue-specific mRNA expression, or of serumand tissue protein levels in transgenic animals before, during, andafter challenge with experimental drug therapies.

[0132] Embryonic Stem Cells

[0133] Embryonic (ES) stem cells isolated from rodent embryos retain thepotential to form embryonic tissues. When ES cells are placed inside acarrier embryo, they resume normal development and contribute to tissuesof the live-born animal. ES cells are the preferred cells used in thecreation of experimental knockout and knockin rodent strains. Mouse EScells, such as the mouse 129/SvJ cell line, are derived from the earlymouse embryo and are grown under culture conditions well known in theart. Vectors used to produce a transgenic strain contain a disease genecandidate and a marker gen, the latter serves to identify the presenceof the introduced disease gene. The vector is transformed into ES cellsby methods well known in the art, and transformed ES cells areidentified and microinjected into mouse cell blastocysts such as thosefrom the C57BL/6 mouse strain. The blastocysts are surgicallytransferred to pseudopregnant dams, and the resulting chimeric progenyare genotyped and bred to produce heterozygous or homozygous strains.

[0134] ES cells derived from human blastocysts may be manipulated invitro to differentiate into at least eight separate cell lineages. Theselineages are used to study the differentiation of various cell types andtissues in vitro, and they include endoderm, mesoderm, and ectodermalcell types which differentiate into, for example, neural cells,hematopoietic lineages, and cardiomyocytes.

[0135] Knockout Analysis

[0136] In gene knockout analysis, a region of a mammalian gene isenzymatically modified to include a non-mammalian gene such as theneomycin phosphotransferase gene (neo; Capecchi (1989) Science244:1288-1292). The modified gene is transformed into cultured ES cellsand integrates into the endogenous genome by homologous recombination.The inserted sequence disrupts transcription and translation of theendogenous gene. Transformed cells are injected into rodent blastulae,and the blastulae are implanted into pseudopregnant dams. Transgenicprogeny are crossbred to obtain homozygous inbred lines which lack afunctional copy of the mammalian gene. In one example, the mammaliangene is a human gene.

[0137] Knockin Analysis

[0138] ES cells can be used to create knockin humanized animals (pigs)or transgenic animal models (mice or rats) of human diseases. Withknockin technology, a region of a human gene is injected into animal EScells, and the human sequence integrates into the animal cell genome.Transformed cells are injected into blastulae and the blastulae areimplanted as described above. Transgenic progeny or inbred lines arestudied and treated with potential pharmaceutical agents to obtaininformation on treatment of the analogous human condition. These methodshave been used to model several human diseases.

[0139] Non-Human Primate Model

[0140] The field of animal testing deals with data and methodology frombasic sciences such as physiology, genetics, chemistry, pharmacology andstatistics. These data are paramount in evaluating the effects oftherapeutic agents on non-human primates as they can be related to humanhealth. Monkeys are used as human surrogates in vaccine and drugevaluations, and their responses are relevant to human exposures undersimilar conditions. Cynomolgus and Rhesus monkeys (Macaca fascicularisand Macaca mulatta, respectively) and Common Marmosets (Callithrixjacchus) are the most common non-human primates (NHPs) used in theseinvestigations. Since great cost is associated with developing andmaintaining a colony of NHPs, early research and toxicological studiesare usually carried out in rodent models. In studies using behavioralmeasures such as drug addiction, NHPs are the first choice test animal.In addition, NHPs and individual humans exhibit differentialsensitivities to many drugs and toxins and can be classified as a rangeof phenotypes from “extensive metabolizers” to “poor metabolizers” ofthese agents.

[0141] In additional embodiments, the cDNAs which encode the mammalianprotein may be used in any molecular biology techniques that have yet tobe developed, provided the new techniques rely on properties of cDNAsthat are currently known, including, but not limited to, such propertiesas the triplet genetic code and specific base pair interactions.

EXAMPLES

[0142] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention. For purposes of example, preparation of the human tumorousadrenal tissue library (ADRETUT06) will be described.

[0143] I cDNA Library Construction

[0144] The ADRETUT06 cDNA library was constructed from tumorous adrenaltissue obtained from a 57-year-old Caucasian female during an unilateraladrenalectomy. The frozen tissue was homogenized and lysed in TRIZOLreagent (1 g tissue/10 ml; Life Technologies) using a POLYTRONhomogenizer (Brinkmann Instruments, Westbury N.J.). After a briefincubation on ice, chloroform was added (1:5 v/v) and the lysate wascentrifuged. The upper chloroform layer was removed, the aqueous phasetransferred to a fresh tube and the RNA precipitated with isopropanol,resuspended in DEPC-treated water, and treated with DNAse for 25 min at37 C. Extraction and precipitation were repeated as before. The mRNA wasthen isolated using the OLIGOTEX kit (Qiagen, Chatsworth Calif.) andused to construct the cDNA library.

[0145] The mRNA was handled according to the recommended protocols inthe SUPERSCRIPT plasmid system (Life Technologies) which contains a NotIprimer-adaptor designed to prime the first strand cDNA synthesis at thepoly(A) tail of mRNAs. Double stranded cDNA was blunted, ligated toEcoRI adaptors and digested with NotI (New England Biolabs, BeverlyMass.). The cDNAs were fractionated on a SEPHAROSE CL4B column (APB),and those cDNAs exceeding 400 bp were ligated into pINCY plasmid (IncyteGenomics). The plasmid pINCY was subsequently transformed into DH5αcompetent cells (Life Technologies).

[0146] II Construction of pINCY Plasmid

[0147] The plasmid was constructed by digesting the PSPORT1 plasmid(Life Technologies) with EcoRI restriction enzyme (New England Biolabs,Beverly Mass.) and filling the overhanging ends using Klenow enzyme (NewEngland Biolabs) and 2′-deoxynucleotide 5′-triphosphates (dNTPs). Theplasmid was self-ligated and transformed into the bacterial host, E.coli strain JM109.

[0148] An intermediate plasmid produced by the bacteria (pSPORT 1-ΔRI)showed no digestion with EcoRI and was digested with Hind III (NewEngland Biolabs) and the overhanging ends were again filled in withKlenow and dNTPs. A linker sequence was phosphorylated, ligated onto the5′ blunt end, digested with EcoRI, and self-ligated. Followingtransformation into JM109 host cells, plasmids were isolated and testedfor preferential digestibility with EcoRI, but not with Hind III. Asingle colony that met this criteria was designated pINCY plasmid.

[0149] After testing the plasmid for its ability to incorporate cDNAsfrom a library prepared using NotI and EcoRI restriction enzymes,several clones were sequenced; and a single clone containing an insertof approximately 0.8 kb was selected from which to prepare a largequantity of the plasmid. After digestion with NotI and EcoRI, theplasmid was isolated on an agarose gel and purified using a QIAQUICKcolumn (Qiagen) for use in library construction.

[0150] III Isolation and Sequencing of cDNA Clones

[0151] Plasmid DNA was released from the cells and purified using eitherthe MINIPREP kit (Edge Biosystems, Gaithersburg Md.) or the REAL PREP 96plasmid kit (Qiagen). This kit consists of a 96-well block with reagentsfor 960 purifications. The recommended protocol was employed except forthe following changes: 1) the bacteria were cultured in 1 ml of sterileTERRIFIC BROTH (BD Biosciences, Sparks Md.) with carbenicillin (carb.)at 25 mg/l and glycerol at 0.4%; 2) after inoculation, the cells werecultured for 19 hours and then lysed with 0.3 ml of lysis buffer; and 3)following isopropanol precipitation, the plasmid DNA pellet wasresuspended in 0.1 ml of distilled water. After the last step in theprotocol, samples were transferred to a 96-well block for storage at 4C.

[0152] The cDNAs were prepared for sequencing using the MICROLAB 2200system (Hamilton) in combination with the DNA ENGINE thermal cyclers (MJResearch). The cDNAs were sequenced by the method of Sanger and Coulson(1975; J Mol Biol 94:441-448) using an ABI PRISM 377 sequencing system(Applied Biosystems) or the MEGABACE 1000 DNA sequencing system (APB).Most of the isolates were sequenced according to standard ABI protocolsand kits (Applied Biosystems) with solution volumes of 0.25×-1.0×concentrations. In the alternative, cDNAs were sequenced using solutionsand dyes from APB.

[0153] IV Extension of cDNA Sequences

[0154] The cDNAs were extended using the cDNA clone and oligonucleotideprimers. One primer was synthesized to initiate 5′ extension of theknown fragment, and the other, to initiate 3′ extension of the knownfragment. The initial primers were designed using OLIGO primer analysissoftware (Molecular Biology Insights), to be about 22 to 30 nucleotidesin length, to have a GC content of about 50% or more, and to anneal tothe target sequence at temperatures of about 68 C. to about 72 C. Anystretch of nucleotides that would result in hairpin structures andprimer-primer dimerizations was avoided.

[0155] Selected cDNA libraries were used as templates to extend thesequence. If more than one extension was necessary, additional or nestedsets of primers were designed. Preferred libraries have beensize-selected to include larger cDNAs and random primed to contain moresequences with 5′ or upstream regions of genes. Genomic libraries areused to obtain regulatory elements, especially extension into the 5′promoter binding region.

[0156] High fidelity amplification was obtained by PCR using methodssuch as that taught in U.S. Pat. No. 5,932,451. PCR was performed in96-well plates using the DNA ENGINE thermal cycler (MJ Research). Thereaction mix contained DNA template, 200 nmol of each primer, reactionbuffer containing Mg²⁺, (NH₄)₂SO₄, and β-mercaptoethanol, Taq DNApolymerase (APB), ELONGASE enzyme (Life Technologies), and Pfu DNApolymerase (Stratagene), with the following parameters for primer pairPCI A and PCI B (Incyte Genomics): Step 1: 94 C., three min; Step 2: 94C., 15 sec; Step 3: 60 C., one min; Step 4: 68 C., two min; Step 5:Steps 2, 3, and 4 repeated 20 times; Step 6: 68 C., five min; Step 7:storage at 4 C. In the alternative, the parameters for primer pair T7and SK+ (Stratagene) were as follows: Step 1: 94 C., three min; Step 2:94 C., 15 sec; Step 3: 57 C., one min; Step 4: 68 C., two min; Step 5:Steps 2, 3, and 4 repeated 20 times; Step 6: 68 C., five min; Step 7:storage at 4 C.

[0157] The concentration of DNA in each well was determined bydispensing 100 μl PICOGREEN quantitation reagent (0.25% reagent in 1×TE, v/v; Molecular Probes) and 0.5 μl of undiluted PCR product into eachwell of an opaque fluorimeter plate (Corning, Acton Mass.) and allowingthe DNA to bind to the reagent. The plate was scanned in a Fluoroskan II(Labsystems Oy) to measure the fluorescence of the sample and toquantify the concentration of DNA. A 5 μl to 10 μl aliquot of thereaction mixture was analyzed by electrophoresis on a 1% agarosemini-gel to determine which reactions were successful in extending thesequence.

[0158] The extended clones were desalted, concentrated, transferred to384-well plates, digested with CviJI cholera virus endonuclease(Molecular Biology Research, Madison Wis.), and sonicated or shearedprior to religation into pUC18 vector (APB). For shotgun sequences, thedigested nucleotide sequences were separated on low concentration (0.6to 0.8%) agarose gels, fragments were excised, and the agar was digestedwith AGARACE enzyme (Promega). Extended clones were religated using T4DNA ligase (New England Biolabs) into pUC18 vector (APB), treated withPfu DNA polymerase (Stratagene) to fill-in restriction site overhangs,and transfected into E. coli competent cells. Transformed cells wereselected on antibiotic-containing media, and individual colonies werepicked and cultured overnight at 37 C. in 384-well plates in LB/2×carbenicillin liquid media.

[0159] The cells were lysed, and DNA was amplified using primers, TaqDNA polymerase (APB) and Pfu DNA polymerase (Stratagene) with thefollowing parameters: Step 1: 94 C., three min; Step 2: 94 C., 15 sec;Step 3: 60 C., one min; Step 4: 72 C., two min; Step 5: steps 2, 3, and4 repeated 29 times; Step 6: 72 C., five min; Step 7: storage at 4 C.DNA was quantified using PICOGREEN quantitative reagent (MolecularProbes) as described above. Samples with low DNA recoveries werereamplified using the conditions described above. Samples were dilutedwith 20% dimethylsulfoxide (DMSO; 1:2, v/v), and sequenced usingDYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECTcycle sequencing kit (APB) or the ABI PRISM BIGDYE terminator cyclesequencing kit (Applied Biosystems).

[0160] V Homology Searching of cDNA Clones and Their Deduced Proteins

[0161] The cDNAs of the Sequence Listing or their deduced amino acidsequences were used to query databases such as GenBank, SwissProt,BLOCKS, and the like. These databases that contain previously identifiedand annotated sequences or domains were searched using BLAST or BLAST 2(Altschul et al. supra; Altschul, supra) to produce alignments and todetermine which sequences were exact matches or homologs. The alignmentswere to sequences of prokaryotic (bacterial) or eukaryotic (animal,fungal, or plant) origin. Alternatively, algorithms such as the onedescribed in Smith and Smith (1992, Protein Engineering 5:35-51) couldhave been used to deal with primary sequence patterns and secondarystructure gap penalties. All of the sequences disclosed in thisapplication have lengths of at least 49 nucleotides, and no more than12% uncalled bases (where N is recorded rather than A, C, G, or T).

[0162] As detailed in Karlin (supra), BLAST matches between a querysequence and a database sequence were evaluated statistically and onlyreported when they satisfied the threshold of 10⁻²⁵ for nucleotides and10⁻¹⁴ for peptides. Homology was also evaluated by product scorecalculated as follows: the % nucleotide or amino acid identity [betweenthe query and reference sequences] in BLAST is multiplied by the %maximum possible BLAST score [based on the lengths of query andreference sequences] and then divided by 100. In comparison withhybridization procedures used in the laboratory, the electronicstringency for an exact match was set at 70, and the conservative lowerlimit for an exact match was set at approximately 40 (with 1-2% errordue to uncalled bases).

[0163] The BLAST software suite, freely available sequence comparisonalgorithms (NCBI, Bethesda Md.;http://www.ncbi.nlm.nih.gov/gorf/bl2.html), includes various sequenceanalysis programs including “blastn” that is used to align nucleic acidmolecules and BLAST 2 that is used for direct pairwise comparison ofeither nucleic or amino acid molecules. BLAST programs are commonly usedwith gap and other parameters set to default settings, e.g.: Matrix:BLOSUM62; Reward for match: 1; Penalty for mismatch: −2; Open Gap: 5 andExtension Gap: 2 penalties; Gap×drop-off: 50; Expect: 10; Word Size: 11;and Filter: on. Identity is measured over the entire length of asequence or some smaller portion thereof. Brenner et al. (1998; ProcNatl Acad Sci 95:6073-6078, incorporated herein by reference) analyzedthe BLAST for its ability to identify structural homologs by sequenceidentity and found 30% identity is a reliable threshold for sequencealignments of at least 150 residues and 40%, for alignments of at least70 residues.

[0164] The mammalian cDNAs of this application were compared withassembled consensus sequences or templates found in the LIFESEQ GOLDdatabase. Component sequences from cDNA, extension, full length, andshotgun sequencing projects were subjected to PHRED analysis andassigned a quality score. All sequences with an acceptable quality scorewere subjected to various pre-processing and editing pathways to removelow quality 3′ ends, vector and linker sequences, polyA tails, Alurepeats, mitochondrial and ribosomal sequences, and bacterialcontamination sequences. Edited sequences had to be at least 50 bp inlength, and low-information sequences and repetitive elements such asdinucleotide repeats, Alu repeats, and the like, were replaced by “Ns”or masked.

[0165] Edited sequences were subjected to assembly procedures in whichthe sequences were assigned to gene bins. Each sequence could onlybelong to one bin, and sequences in each bin were assembled to produce atemplate. Newly sequenced components were added to existing bins usingBLAST and CROSSMATCH. To be added to a bin, the component sequences hadto have a BLAST quality score greater than or equal to 150 and analignment of at least 82% local identity. The sequences in each bin wereassembled using PHRAP. Bins with several overlapping component sequenceswere assembled using DEEP PHRAP. The orientation of each template wasdetermined based on the number and orientation of its componentsequences.

[0166] Bins were compared to one another and those having localsimilarity of at least 82% were combined and reassembled. Bins havingtemplates with less than 95% local identity were split. Templates weresubjected to analysis by STITCHER/EXON MAPPER algorithms that analyzethe probabilities of the presence of splice variants, alternativelyspliced exons, splice junctions, differential expression of alternativespliced genes across tissue types or disease states, and the like.Assembly procedures were repeated periodically, and templates wereannotated using BLAST against GenBank databases such as GBpri. An exactmatch was defined as having from 95% local identity over 200 base pairsthrough 100% local identity over 100 base pairs and a homolog match ashaving an E-value (or probability score) of ≦1×10⁻⁸. The templates werealso subjected to frameshift FAST× against GENPEPT, and homolog matchwas defined as having an E-value of ≦1×10⁻⁸. Template analysis andassembly was described in U.S. Ser. No. 09/276,534, filed Mar. 25, 1999.

[0167] Following assembly, templates were subjected to BLAST, motif, andother functional analyses and categorized in protein hierarchies usingmethods described in U.S. Ser. No. 08/812,290 and U.S. Ser. No.08/811,758, both filed Mar. 6, 1997; in U.S. Ser. No. 08/947,845, filedOct. 9, 1997; and in U.S. Ser. No. 09/034,807, filed Mar. 4, 1998. Thentemplates were analyzed by translating each template in all threeforward reading frames and searching each translation against the PFAMdatabase of hidden Markov model-based protein families and domains usingthe HMMER software package (Washington University School of Medicine,St. Louis Mo.; http://pfam.wustl.edu/). The cDNA was further analyzedusing MACDNASIS PRO software (Hitachi Software Engineering), andLASERGENE software (DNASTAR) and queried against public databases suchas the GenBank rodent, mammalian, vertebrate, prokaryote, and eukaryotedatabases, SwissProt, BLOCKS, PRINTS, PFAM, and Prosite.

[0168] VI Chromosome Mapping

[0169] Radiation hybrid and genetic mapping data available from publicresources such as the Stanford Human Genome Center (SHGC), WhiteheadInstitute for Genome Research (WIGR), and Genethon are used to determineif any of the cDNAs presented in the Sequence Listing have been mapped.Any of the fragments of the cDNA encoding VMP that have been mappedresult in the assignment of all related regulatory and coding sequencesmapping to the same location. The genetic map locations are described asranges, or intervals, of human chromosomes. The map position of aninterval, in cM (which is roughly equivalent to 1 megabase of humanDNA), is measured relative to the terminus of the chromosomal p-arm.

[0170] VII Hybridization Technologies and Analyses

[0171] Immobilization of cDNAs on a Substrate

[0172] The cDNAs are applied to a substrate by one of the followingmethods. A mixture of cDNAs is fractionated by gel electrophoresis andtransferred to a nylon membrane by capillary transfer. Alternatively,the cDNAs are individually ligated to a vector and inserted intobacterial host cells to form a library. The cDNAs are then arranged on asubstrate by one of the following methods. In the first method,bacterial cells containing individual clones are robotically picked andarranged on a nylon membrane. The membrane is placed on LB agarcontaining selective agent (carbenicillin, kanamycin, ampicillin, orchloramphenicol depending on the vector used) and incubated at 37 C. for16 hr. The membrane is removed from the agar and consecutively placedcolony side up in 10% SDS, denaturing solution (1.5 M NaCl, 0.5 M NaOH),neutralizing solution (1.5 M NaCl, 1 M Tris, pH 8.0), and twice in 2×SSCfor 10 min each. The membrane is then UV irradiated in a STRATALINKERUV-crosslinker (Stratagene).

[0173] In the second method, cDNAs are amplified from bacterial vectorsby thirty cycles of PCR using primers complementary to vector sequencesflanking the insert. PCR amplification increases a startingconcentration of 1-2 ng nucleic acid to a final quantity greater than 5μg. Amplified nucleic acids from about 400 bp to about 5000 bp in lengthare purified using SEPHACRYL-400 beads (APB). Purified nucleic acids arearranged on a nylon membrane manually or using a dot/slot blottingmanifold and suction device and are immobilized by denaturation,neutralization, and UV irradiation as described above. Purified nucleicacids are robotically arranged and immobilized on polymer-coated glassslides using the procedure described in U.S. Pat. No. 5,807,522.Polymer-coated slides are prepared by cleaning glass microscope slides(Corning, Acton Mass.) by ultrasound in 0.1% SDS and acetone, etching in4% hydrofluoric acid (VWR Scientific Products, West Chester Pa.),coating with 0.05% aminopropyl silane (Sigma Aldrich) in 95% ethanol,and curing in a 110 C. oven. The slides are washed extensively withdistilled water between and after treatments. The nucleic acids arearranged on the slide and then immobilized by exposing the array to UVirradiation using a STRATALINKER UV-crosslinker (Stratagene). Arrays arethen washed at room temperature in 0.2% SDS and rinsed three times indistilled water. Non-specific binding sites are blocked by incubation ofarrays in 0.2% casein in phosphate buffered saline (PBS; Tropix, BedfordMass.) for 30 min at 60 C.; then the arrays are washed in 0.2% SDS andrinsed in distilled water as before.

[0174] Probe Preparation for Membrane Hybridization

[0175] Hybridization probes derived from the cDNAs of the SequenceListing are employed for screening cDNAs, mRNAs, or genomic DNA inmembrane-based hybridizations. Probes are prepared by diluting the cDNAsto a concentration of 40-50 ng in 45 μl TE buffer, denaturing by heatingto 100 C. for five min, and briefly centrifuging. The denatured cDNA isthen added to a REDIPRIME tube (APB), gently mixed until blue color isevenly distributed, and briefly centrifuged. Five μl of [³²P]dCTP isadded to the tube, and the contents are incubated at 37 C. for 10 min.The labeling reaction is stopped by adding 5 μl of 0.2M EDTA, and probeis purified from unincorporated nucleotides using a PROBEQUANT G-50microcolumn (APB). The purified probe is heated to 100 C. for five min,snap cooled for two min on ice, and used in membrane-basedhybridizations as described below.

[0176] Probe Preparation for Polymer Coated Slide Hybridization

[0177] Hybridization probes derived from mRNA isolated from samples areemployed for screening cDNAs of the Sequence Listing in array-basedhybridizations. Probe is prepared using the GEMbright kit (IncyteGenomics) by diluting mRNA to a concentration of 200 ng in 9 μl TEbuffer and adding 5 μl 5× buffer, 1 μl 0.1 M DTT, 3 μl Cy3 or Cy5labeling mix, 1 μl RNase inhibitor, 1 μl reverse transcriptase, and 5 μl1× yeast control mRNAs. Yeast control mRNAs are synthesized by in vitrotranscription from noncoding yeast genomic DNA (W. Lei, unpublished). Asquantitative controls, one set of control mRNAs at 0.002 ng, 0.02 ng,0.2 ng, and 2 ng are diluted into reverse transcription reaction mixtureat ratios of 1:100,000, 1:10,000, 1:1000, and 1:100 (w/w) to sample mRNArespectively. To examine mRNA differential expression patterns, a secondset of control mRNAs are diluted into reverse transcription reactionmixture at ratios of 1:3, 3:1, 1:10, 10:1, 1:25, and 25:1 (w/w). Thereaction mixture is mixed and incubated at 37 C. for two hr. Thereaction mixture is then incubated for 20 min at 85 C., and probes arepurified using two successive CHROMA SPIN+TE 30 columns (Clontech, PaloAlto Calif.). Purified probe is ethanol precipitated by diluting probeto 90 μl in DEPC-treated water, adding 2 μl 1 mg/ml glycogen, 60 μl 5 Msodium acetate, and 300 μl 100% ethanol. The probe is centrifuged for 20min at 20,800×g, and the pellet is resuspended in 12 μl resuspensionbuffer, heated to 65 C. for five min, and mixed thoroughly. The probe isheated and mixed as before and then stored on ice. Probe is used in highdensity array-based hybridizations as described below.

[0178] Membrane-Based Hybridization

[0179] Membranes are pre-hybridized in hybridization solution containing1% Sarkosyl and 1× high phosphate buffer (0.5 M NaCl, 0.1 M Na₂HPO₄, 5mM EDTA, pH 7) at 55 C. for two hr. The probe, diluted in 15 ml freshhybridization solution, is then added to the membrane. The membrane ishybridized with the probe at 55 C. for 16 hr. Following hybridization,the membrane is washed for 15 min at 25 C. in 1 mM Tris (pH 8.0), 1%Sarkosyl, and four times for 15 min each at 25 C. in 1 mM Tris (pH 8.0).To detect hybridization complexes, XOMAT-AR film (Eastman Kodak,Rochester N.Y.) is exposed to the membrane overnight at −70 C.,developed, and examined visually.

[0180] Polymer Coated Slide-Based Hybridization

[0181] Probe is heated to 65 C. for five min, centrifuged five min at9400 rpm in a 5415C microcentrifuge (Eppendorf Scientific, WestburyN.Y.), and then 18 μl is aliquoted onto the array surface and coveredwith a coverslip. The arrays are transferred to a waterproof chamberhaving a cavity just slightly larger than a microscope slide. Thechamber is kept at 100% humidity internally by the addition of 140 μl of5×SSC in a corner of the chamber. The chamber containing the arrays isincubated for about 6.5 hr at 60 C. The arrays are washed for 10 min at45 C. in 1×SSC, 0.1% SDS, and three times for 10 min each at 45 C. in0.1×SSC, and dried.

[0182] Hybridization reactions are performed in absolute or differentialhybridization formats. In the absolute hybridization format, probe fromone sample is hybridized to array elements, and signals are detectedafter hybridization complexes form. Signal strength correlates withprobe mRNA levels in the sample. In the differential hybridizationformat, differential expression of a set of genes in two biologicalsamples is analyzed. Probes from the two samples are prepared andlabeled with different labeling moieties. A mixture of the two labeledprobes is hybridized to the array elements, and signals are examinedunder conditions in which the emissions from the two different labelsare individually detectable. Elements on the array that are hybridizedto substantially equal numbers of probes derived from both biologicalsamples give a distinct combined fluorescence (Shalon WO95/35505).

[0183] Hybridization complexes are detected with a microscope equippedwith an Innova 70 mixed gas 10 W laser (Coherent, Santa Clara Calif.)capable of generating spectral lines at 488 nm for excitation of Cy3 andat 632 nm for excitation of Cy5. The excitation laser light is focusedon the array using a 20× microscope objective (Nikon, Melville N.Y.).The slide containing the array is placed on a computer-controlled X-Ystage on the microscope and raster-scanned past the objective with aresolution of 20 micrometers. In the differential hybridization format,the two fluorophores are sequentially excited by the laser. Emittedlight is split, based on wavelength, into two photomultiplier tubedetectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater N.J.)corresponding to the two fluorophores. Appropriate filters positionedbetween the array and the photomultiplier tubes are used to filter thesignals. The emission maxima of the fluorophores used are 565 nm for Cy3and 650 nm for Cy5. The sensitivity of the scans is calibrated using thesignal intensity generated by the yeast control mRNAs added to the probemix. A specific location on the array contains a complementary DNAsequence, allowing the intensity of the signal at that location to becorrelated with a weight ratio of hybridizing species of 1:100,000.

[0184] The output of the photomultiplier tube is digitized using a12-bit RTI-835H analog-to-digital (A/D) conversion board (AnalogDevices, Norwood Mass.) installed in an IBM-compatible PC computer. Thedigitized data are displayed as an image where the signal intensity ismapped using a linear 20-color transformation to a pseudocolor scaleranging from blue (low signal) to red (high signal). The data is alsoanalyzed quantitatively. Where two different fluorophores are excitedand measured simultaneously, the data are first corrected for opticalcrosstalk (due to overlapping emission spectra) between the fluorophoresusing the emission spectrum for each fluorophore. A grid is superimposedover the fluorescence signal image such that the signal from each spotis centered in each element of the grid. The fluorescence signal withineach element is then integrated to obtain a numerical valuecorresponding to the average intensity of the signal. The software usedfor signal analysis is the GEMTOOLS program (Incyte Genomics).

[0185] VIII Electronic Analysis

[0186] BLAST was used to search for identical or related molecules inthe GenBank or LIFESEQ databases (Incyte Genomics). The product scorefor human and rat sequences was calculated as follows: the BLAST scoreis multiplied by the % nucleotide identity and the product is divided by(5 times the length of the shorter of the two sequences), such that a100% alignment over the length of the shorter sequence gives a productscore of 100. The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1% to 2% error, and with a product score of at least 70, the matchwill be exact. Similar or related molecules are usually identified byselecting those which show product scores between 8 and 40.

[0187] Northern analysis was performed at a product score of 70 as shownin Tables 1, 2, 3, and 4. All sequences and cDNA libraries in theLIFESEQ database were categorized by system, organ/tissue and cell type.The categories included cardiovascular system, connective tissue,digestive system, embryonic structures, endocrine system, exocrineglands, female and male genitalia, germ cells, hemic/immune system,liver, musculoskeletal system, nervous system, pancreas, respiratorysystem, sense organs, skin, stomatognathic system, unclassified/mixed,and the urinary tract. For each category, the number of libraries inwhich the sequence was expressed were counted and shown over the totalnumber of libraries in that category. In a non-normalized library,expression levels of two or more are significant.

[0188] IX Complementary Molecules

[0189] Molecules complementary to the cDNA, from about 5 (PNA) to about5000 bp (complement of a cDNA insert), are used to detect or inhibitgene expression. These molecules are selected using OLIGO primeranalysis software (Molecular Biology Insights). Detection is describedin Example VII. To inhibit transcription by preventing promoter binding,the complementary molecule is designed to bind to the most unique 5′sequence and includes nucleotides of the 5′ UTR upstream of theinitiation codon of the open reading frame. Complementary moleculesinclude genomic sequences (such as enhancers or introns) and are used in“triple helix” base pairing to compromise the ability of the doublehelix to open sufficiently for the binding of polymerases, transcriptionfactors, or regulatory molecules. To inhibit translation, acomplementary molecule is designed to prevent ribosomal binding to themRNA encoding the mammalian protein.

[0190] Complementary molecules are placed in expression vectors and usedto transform a cell line to test efficacy; into an organ, tumor,synovial cavity, or the vascular system for transient or short termtherapy; or into a stem cell, zygote, or other reproducing lineage forlong term or stable gene therapy. Transient expression lasts for a monthor more with a non-replicating vector and for three months or more ifappropriate elements for inducing vector replication are used in thetransformation/expression system.

[0191] Stable transformation of appropriate dividing cells with a vectorencoding the complementary molecule produces a transgenic cell line,tissue, or organism (U.S. Pat. No. 4,736,866). Those cells thatassimilate and replicate sufficient quantities of the vector to allowstable integration also produce enough complementary molecules tocompromise or entirely eliminate activity of the cDNA encoding themammalian protein.

[0192] X Expression of VMP

[0193] Expression and purification of the mammalian protein are achievedusing either a mammalian cell expression system or an insect cellexpression system. The pUB6/V5-His vector system (Invitrogen, CarlsbadCalif.) is used to express VMP in CHO cells. The vector contains theselectable bsd gene, multiple cloning sites, the promoter/enhancersequence from the human ubiquitin C gene, a C-terminal V5 epitope forantibody detection with anti-V5 antibodies, and a C-terminalpolyhistidine (6xHis) sequence for rapid purification on PROBOND resin(Invitrogen). Transformed cells are selected on media containingblasticidin.

[0194]Spodoptera frugiperda (Sf9) insect cells are infected withrecombinant Autographica californica nuclear polyhedrosis virus(baculovirus). The polyhedrin gene is replaced with the mammalian cDNAby homologous recombination and the polyhedrin promoter drives cDNAtranscription. The protein is synthesized as a fusion protein with 6xhiswhich enables purification as described above. Purified protein is usedin the following activity and to make antibodies

[0195] XI Production of Antibodies

[0196] VMP is purified using polyacrylamide gel electrophoresis and usedto immunize mice or rabbits. Antibodies are produced using the protocolsbelow. Alternatively, the amino acid sequence of VMP is analyzed usingLASERGENE software (DNASTAR) to determine regions of high antigenicity.An antigenic epitope, usually found near the C-terminus or in ahydrophilic region is selected, synthesized, and used to raiseantibodies. Typically, epitopes of about 15 residues in length areproduced using an ABI 431A peptide synthesizer (Applied Biosystems)using Fmoc-chemistry and coupled to KLH (Sigma-Aldrich) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester to increase antigenicity.

[0197] Rabbits are immunized with the epitope-KLH complex in completeFreund's adjuvant. Immunizations are repeated at intervals thereafter inincomplete Freund's adjuvant. After a minimum of seven weeks for mouseor twelve weeks for rabbit, antisera are drawn and tested forantipeptide activity. Testing involves binding the peptide to plastic,blocking with 1% bovine serum albumin, reacting with rabbit antisera,washing, and reacting with radio-iodinated goat anti-rabbit IgG. Methodswell known in the art are used to determine antibody titer and theamount of complex formation.

[0198] XII Purification of Naturally Occurring Protein Using SpecificAntibodies

[0199] Naturally occurring or recombinant protein is purified byimmunoaffinity chromatography using antibodies which specifically bindthe protein. An immunoaffinity column is constructed by covalentlycoupling the antibody to CNBr-activated SEPHAROSE resin (APB). Mediacontaining the protein is passed over the immunoaffinity column, and thecolumn is washed using high ionic strength buffers in the presence ofdetergent to allow preferential absorbance of the protein. Aftercoupling, the protein is eluted from the column using a buffer of pH 2-3or a high concentration of urea or thiocyanate ion to disruptantibody/protein binding, and the protein is collected.

[0200] XIII Screening Molecules for Specific Binding with the cDNA orProtein

[0201] The cDNA, or fragments thereof, or the protein, or portionsthereof, are labeled with ³²P-dCTP, Cy3-dCTP, or Cy5-dCTP (APB), or withBIODIPY or FITC (Molecular Probes, Eugene Oreg.), respectively.Libraries of candidate molecules or compounds previously arranged on asubstrate are incubated in the presence of labeled cDNA or protein.After incubation under conditions for either a nucleic acid or aminoacid sequence, the substrate is washed, and any position on thesubstrate retaining label, which indicates specific binding or complexformation, is assayed, and the ligand is identified. Data obtained usingdifferent concentrations of the nucleic acid or protein are used tocalculate affinity between the labeled nucleic acid or protein and thebound molecule.

[0202] XIV Two-Hybrid Screen

[0203] A yeast two-hybrid system, MATCHMAKER LexA Two-Hybrid system(Clontech Laboratories, Palo Alto Calif.), is used to screen forpeptides that bind the mammalian protein of the invention. A cDNAencoding the protein is inserted into the multiple cloning site of apLexA vector, ligated, and transformed into E. coli. cDNA, prepared frommRNA, is inserted into the multiple cloning site of a pB42AD vector,ligated, and transformed into E. coli to construct a cDNA library. ThepLexA plasmid and pB42AD-cDNA library constructs are isolated from E.coli and used in a 2:1 ratio to co-transform competent yeastEGY48[p8op-lacZ] cells using a polyethylene glycol/lithium acetateprotocol. Transformed yeast cells are plated on synthetic dropout (SD)media lacking histidine (-His), tryptophan (-Trp), and uracil (-Ura),and incubated at 30 C. until the colonies have grown up and are counted.The colonies are pooled in a minimal volume of 1× TE (pH 7.5), replatedon SD/-His/-Leu/-Trp/-Ura media supplemented with 2% galactose (Gal), 1%raffinose (Raf), and 80 mg/ml 5-bromo-4-chloro-3-indolylβ-d-galactopyranoside (X-Gal), and subsequently examined for growth ofblue colonies. Interaction between expressed protein and cDNA fusionproteins activates expression of a LEU2 reporter gene in EGY48 andproduces colony growth on media lacking leucine (-Leu). Interaction alsoactivates expression of β-galactosidase from the p8op-lacZ reporterconstruct that produces blue color in colonies grown on X-Gal.

[0204] Positive interactions between expressed protein and cDNA fusionproteins are verified by isolating individual positive colonies andgrowing them in SD/-Trp/-Ura liquid medium for 1 to 2 days at 30 C. Asample of the culture is plated on SD/-Trp/-Ura media and incubated at30 C. until colonies appear. The sample is replica-plated onSD/-Trp/-Ura and SD/-His/-Trp/-Ura plates. Colonies that grow on SDcontaining histidine but not on media lacking histidine have lost thepLexA plasmid. Histidine-requiring colonies are grown onSD/Gal/Raf/X-Gal/-Trp/-Ura, and white colonies are isolated andpropagated. The pB42AD-cDNA plasmid, which contains a cDNA encoding aprotein that physically interacts with the mammalian protein, isisolated from the yeast cells and characterized.

[0205] XV VMP Assay

[0206] VMP or biologically active fragments thereof are labeled with¹²⁵I Bolton-Hunter reagent (Bolton et al. (1973) Biochem J 133:529-539).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled VMP, washed and any wells withlabeled VMP complex are assayed. Data obtained using differentconcentrations of VMP are used to calculate values for the number,affinity, and association of VMP with the candidate molecules.

[0207] All patents and publications mentioned in the specification areincorporated by reference herein. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in thefield of molecular biology or related fields are intended to be withinthe scope of the following claims. TABLE 1 Clone Abs Pct Tissue CategoryCount Found in Abund Abund Cardiovascular System 266190 25/68  47 0.0177Connective Tissue 144645 21/47  34 0.0235 Digestive System 501101 59/14887 0.0174 Embryonic Structures 106713 7/21 11 0.0103 Endocrine System225386 25/53  61 0.0271 Exocrine Glands 254635 31/64  48 0.0189Reproductive, Female 427284 37/106 60 0.0140 Reproductive, Male 44820732/114 45 0.0100 Germ Cell Tumors 38282 0/5  0 0.0000 Hemic and ImmuneSystem 680277 37/159 62 0.0091 Liver 109378 6/35 7 0.0064Musculoskeletal System 159280 14/47  18 0.0113 Nervous System 95575364/198 128 0.0134 Pancreas 110207 4/24 4 0.0036 Respiratory System390086 39/93  72 0.0185 Sense Organs 19256 0/8  0 0.0000 Skin 72292 3/154 0.0055 Stomatognathic Tumors 12923 0/10 0 0.0000 Unclassified/Mixed120926 1/13 3 0.0025 Urinary Tract 279062 25/64  35 0.0125 Totals5321883 430/1292 726 0.0001

[0208] TABLE 2 Clone Abs Pct Library ID Count Library Description AbundAbund ADRENOT14 3561 adrenal gland, 8M 5 0.1404 THYRTUT03 3626 thyroidtumor, follicular adenoma, 17M 4 0.1103 ADRETUT01 5955 adrenal tumor,mets renal cell CA, 50M 5 0.0840 ADRENOT11 3934 adrenal gland,mw/pheochromocycoma, 43F, m/ADRETUT07 3 0.0763 ADRETUT07 4094 adrenaltumor, pheochromocytoma, 43F, m/ADRENOT11 3 0.0733 ADRETUT05 7872adrenal tumor, pheochromocytoma, 52F 5 0.0635 ADRENOT07 6574 adrenalgland, 61F 4 0.0608 ADRENOT09 3628 adrenal gland, aw/renal cell CA, 43M2 0.0551 ADRENOT08 3981 adrenal gland, 20M 2 0.0502 THYRNOT10 8179thyroid, lymphocytic thyroiditis, mw/papillary CA, 30F 4 0.0489THYRNOT03 7186 thyroid, mw/follicular adenoma, 28F 3 0.0417 THYRNOT087555 thyroid, lymphocytic thyroiditis, mw/papillary CA, 13F 3 0.0397

[0209] TABLE 3 Clone Abs Pct Tissue Category Count Found in Abund AbundCardiovascular System 266190 18/68  27 0.0101 Connective Tissue 14464515/47  18 0.0124 Digestive System 501101 46/148 68 0.0136 EmbryonicStructures 106713 4/21 8 0.0075 Endocrine System 225386 20/53  28 0.0124Exocrine Glands 254635 20/64  32 0.0126 Reproductive, Female 42728430/106 55 0.0129 Reproductive, Male 448207 39/114 68 0.0152 Germ CellTumors 38282 1/5  1 0.0026 Hemic and Immune System 680277 40/159 620.0091 Liver 109378 6/35 7 0.0064 Musculoskeletal System 159280 10/47 11 0.0069 Nervous System 955753 61/198 95 0.0099 Pancreas 110207 6/24 90.0082 Respiratory System 390086 35/93  59 0.0151 Sense Organs 192560/8  0 0.0000 Skin 72292 5/15 6 0.0083 Stomatognathic System 12923 2/103 0.0232 Unclassified/Mixed 120926 4/13 4 0.0033 Urinary Tract 27906217/64  27 0.0097 Totals 5321883 379/1292 588 0.0001

[0210] TABLE 4A Clone Abs Pet Library ID Count Library Description AbundAbund COLNCRT01 2271 colon, Crohn's, mw/benign carcinoid, 40M,m/COLNNOT05 3 0.1321 COLSTUT01 3820 colon tumor, sigmoid, adenoCA, 62M,m/COLNNOT16 5 0.1309 BRSTNOT12 4195 breast, NF breast disease, 32F 40.0954 BRSTTUT25 3491 breast tumor, high vascular density, CA, F 30.0859 BRSTNOT16 4019 breast, papillomatosis, mw/lobular CA, 59F 20.0498 BRSTTUT02 7098 breast tumor, adenoCA, 54F, m/BRSTNOT03 2 0.0282OVARTUTO3 4249 ovary tumor, seroanaplastic CA, 52F 5 0.1177 OVARTUT103608 ovary tumor, mets colon adenoCA, 58F 3 0.0831 OVARNOT09 4295 ovary,follicular cysts, 28F 2 0.0466 CERVNOT01 5006 cervix, cervicitis, 35F 20.0400 UTRSTUP05 15924 uterus tumor, serous papillary CA, F, pooled, 3′CGAP 6 0.0377 OVARTUM02 2932 ovary tumor, papillary serous CA, 64F,WM/WN 1 0.0341 UTRSTUP02 18219 uterus tumor, endometrial adenoCA, F,pooled, 3′ CGAP 6 0.0329 OVARTUP02 3158 ovary tumor, serous papillaryadenoCA, F, 3′ CGAP 1 0.0317 OVARTUP06 6535 ovary tumor, mixed types,pooled, F, 3′ CGAP 2 0.0306 PROSTUP04 865 prostate tumor, cancer, 14,pool, 3′ CGAP 1 0.1156 PROSTMC01 3899 prostate, AH, mw/adenoCA, 55M,m/PROSTUT16, lg cDNA 4 0.1026 PROSDIN01 3427 prostate, AH, mw/adenoCA,66M, NORM, m/PROSTUT10 3 0.0875 PROSNOT16 7368 prostate, AH, mw/adenoCA,68M 4 0.0543 PROSTUT13 3696 prostate tumor, adenoCA, 59M, m/PROSNOT19 20.0541 PROSNOT18 3920 prostate, AH, aw/bladder TC CA, 58M 2 0.0510PROSTMC02 2089 prostate, AH, mw/adenoCA, 48-73M, pool, lg cDNA 1 0.0479PROSTUT18 2201 prostate tumor, adenoCA, 68M, m/PROSTMT03 1 0.0454PROSTUT10 6986 prostate tumor, adenoCA, 66M, m/PROSNOT15, PROSDIN01 30.0429 PROSTUT04 8553 prostate tumor, adenoCA, 57M, m/PROSNOT06 3 0.0351PROSTUT01 3226 prostate tumor, adenoCA, 50M, m/PROSNOT02 1 0.0310PROSTMY01 6509 prostate, AH, mw/adenoCA, node mets, 55M, lg/N,m/PROSTUT16 2 0.0307 PROSTMT03 3821 prostate, mw/adenoCA, 68M,m/PROSTUT18 1 0.0262 PROSNOT06 8829 prostate, AH, mw/adenoCA, 57M,m/PROSTUT04 2 0.0227 PROSNOT15 4140 prostate, AH, mw/adenoCA, 66M,m/PROSTUT10 1 0.0242

[0211] Clone Library ID Count Library Description COLNNOT05 3560 colon,sigmoid, mw/Crohn's, carcinoid, 40M, m/COLNCRT01 BRSTNOT03 6800 breast,PF changes, mw/ductal adenoCA, 54F, m/BRSTTUT02 PROSNOT02 2300 prostate,AH, mw/adenoCA, 50M, m/PROSTUT01 PROSNOT19 3679 prostate, AH,mw/adenoCA, M, m/PROSTUT13, PROSTUS08/19/20/25

[0212]

1 55 1 214 PRT Homo sapiens misc_feature Incyte ID No 743725 1 Met GlyLeu Ala Gly Val Cys Ala Leu Arg Arg Ser Ala Gly Tyr 1 5 10 15 Ile LeuVal Gly Gly Ala Gly Gly Gln Ser Ala Ala Ala Ala Ala 20 25 30 Arg Arg CysSer Glu Gly Glu Trp Ala Ser Gly Gly Val Arg Ser 35 40 45 Phe Ser Arg AlaAla Ala Ala Met Ala Pro Ile Lys Val Gly Asp 50 55 60 Ala Ile Pro Ala ValGlu Val Phe Glu Gly Glu Pro Gly Asn Lys 65 70 75 Val Asn Leu Ala Glu LeuPhe Lys Gly Lys Lys Gly Val Leu Phe 80 85 90 Gly Val Pro Gly Ala Phe ThrPro Gly Cys Ser Lys Thr His Leu 95 100 105 Pro Gly Phe Val Glu Gln AlaGlu Ala Leu Lys Ala Lys Gly Val 110 115 120 Gln Val Val Ala Cys Leu SerVal Asn Asp Ala Phe Val Thr Gly 125 130 135 Glu Trp Gly Arg Ala His LysAla Glu Gly Lys Val Arg Leu Leu 140 145 150 Ala Asp Pro Thr Gly Ala PheGly Lys Glu Thr Asp Leu Leu Leu 155 160 165 Asp Asp Ser Leu Val Ser IlePhe Gly Asn Arg Arg Leu Lys Arg 170 175 180 Phe Ser Met Val Val Gln AspGly Ile Val Lys Ala Leu Asn Val 185 190 195 Glu Pro Asp Gly Thr Gly LeuThr Cys Ser Leu Ala Pro Asn Ile 200 205 210 Ile Ser Gln Leu 2 663 PRTHomo sapiens misc_feature Incyte ID No 2822412 2 Met Ser Ala Arg Leu ProVal Leu Ser Pro Pro Arg Trp Pro Arg 1 5 10 15 Leu Leu Leu Leu Ser LeuLeu Leu Leu Gly Ala Val Pro Gly Pro 20 25 30 Arg Arg Ser Gly Ala Phe TyrLeu Pro Gly Leu Ala Pro Val Asn 35 40 45 Phe Cys Asp Glu Glu Lys Lys SerAsp Glu Cys Lys Ala Glu Ile 50 55 60 Glu Leu Phe Val Asn Arg Leu Asp SerVal Glu Ser Val Leu Pro 65 70 75 Tyr Glu Tyr Thr Ala Phe Asp Phe Cys GlnAla Ser Glu Gly Lys 80 85 90 Arg Pro Ser Glu Asn Leu Gly Gln Val Leu PheGly Glu Arg Ile 95 100 105 Glu Pro Ser Pro Tyr Lys Phe Thr Phe Asn LysLys Glu Thr Cys 110 115 120 Lys Leu Val Cys Thr Lys Thr Tyr His Thr GluLys Ala Glu Asp 125 130 135 Lys Gln Lys Leu Glu Phe Leu Lys Lys Ser MetLeu Leu Asn Tyr 140 145 150 Gln His His Trp Ile Val Asp Asn Met Pro ValThr Trp Cys Tyr 155 160 165 Asp Val Glu Asp Gly Gln Arg Phe Cys Asn ProGly Phe Pro Ile 170 175 180 Gly Cys Tyr Ile Thr Asp Lys Gly His Ala LysAsp Ala Cys Val 185 190 195 Ile Ser Ser Asp Phe His Glu Arg Asp Thr PheTyr Ile Phe Asn 200 205 210 His Val Asp Ile Lys Ile Tyr Tyr His Val ValGlu Thr Gly Ser 215 220 225 Met Gly Ala Arg Leu Val Ala Ala Lys Leu GluPro Lys Ser Phe 230 235 240 Lys His Thr His Ile Asp Lys Pro Asp Cys SerGly Pro Pro Met 245 250 255 Asp Ile Ser Asn Lys Ala Ser Gly Glu Ile LysIle Ala Tyr Thr 260 265 270 Tyr Ser Val Ser Phe Glu Glu Asp Asp Lys IleArg Trp Ala Ser 275 280 285 Arg Trp Asp Tyr Ile Leu Glu Ser Met Pro HisThr His Ile Gln 290 295 300 Trp Phe Ser Ile Met Asn Ser Leu Val Ile ValLeu Phe Leu Ser 305 310 315 Gly Met Val Ala Met Ile Met Leu Arg Thr LeuHis Lys Asp Ile 320 325 330 Ala Arg Tyr Asn Gln Met Asp Ser Thr Glu AspAla Gln Glu Glu 335 340 345 Phe Gly Trp Lys Leu Val His Gly Asp Ile PheArg Pro Pro Arg 350 355 360 Lys Gly Met Leu Leu Ser Val Phe Leu Gly SerGly Thr Gln Ile 365 370 375 Leu Ile Met Thr Phe Val Thr Leu Phe Phe AlaCys Leu Gly Phe 380 385 390 Leu Ser Pro Ala Asn Arg Gly Ala Leu Met ThrCys Ala Val Val 395 400 405 Leu Trp Val Leu Leu Gly Thr Pro Ala Gly TyrVal Ala Ala Arg 410 415 420 Phe Tyr Lys Ser Phe Gly Gly Glu Lys Trp LysThr Asn Val Leu 425 430 435 Leu Thr Ser Phe Leu Cys Pro Gly Ile Val PheAla Asp Phe Phe 440 445 450 Ile Met Asn Leu Ile Leu Trp Gly Glu Gly SerSer Ala Ala Ile 455 460 465 Pro Phe Gly Thr Leu Val Ala Ile Leu Ala LeuTrp Phe Cys Ile 470 475 480 Ser Val Pro Leu Thr Phe Ile Gly Ala Tyr PheGly Phe Lys Lys 485 490 495 Asn Ala Ile Glu His Pro Val Arg Thr Asn GlnIle Pro Arg Gln 500 505 510 Ile Pro Glu Gln Ser Phe Tyr Thr Lys Pro LeuPro Gly Ile Ile 515 520 525 Met Gly Gly Ile Leu Pro Phe Gly Cys Ile PheIle Gln Leu Phe 530 535 540 Phe Ile Leu Asn Ser Ile Trp Ser His Gln MetTyr Tyr Met Phe 545 550 555 Gly Phe Leu Phe Leu Val Phe Ile Ile Leu ValIle Thr Cys Ser 560 565 570 Glu Ala Thr Ile Leu Leu Cys Tyr Phe His LeuCys Ala Glu Asp 575 580 585 Tyr His Trp Gln Trp Arg Ser Phe Leu Thr SerGly Phe Thr Ala 590 595 600 Val Tyr Phe Leu Ile Tyr Ala Val His Tyr PhePhe Ser Lys Leu 605 610 615 Gln Ile Thr Gly Thr Ala Ser Thr Ile Leu TyrPhe Gly Tyr Thr 620 625 630 Met Ile Met Val Leu Ile Phe Phe Leu Phe ThrGly Thr Ile Gly 635 640 645 Phe Phe Ala Cys Phe Trp Phe Val Thr Lys IleTyr Ser Val Val 650 655 660 Lys Val Asp 3 841 DNA Homo sapiensmisc_feature Incyte ID No 743725CB1 3 ggcggcccag gcccgtcttn cgcaggtgtcgccgctgtgc cgctagcggt gccccgnctg 60 ctgcggtggc accagccagg aggcggagtggaagtggccn tggggcgggt atgggactag 120 ctggcgtgtg cgccctgaga cggtnagcgggctatatact cgtcggtggg gccggcggtc 180 agtctgcggc agggcagcaa gacggtgcagtgaaggagag tgggcgtctg gcggggtccg 240 cagtttcagc agagccgctg cagccatggccccaatcaag gtgggagatg ccatcccagc 300 agtggaggtg tttgaagggg agccagggaacaaggtgaac ctggcagagc tgttcaaggg 360 caagaagggt gtgctgtttg gagttcctggggccttcacc cctggatgtt ccaagacaca 420 cctgccaggg tttgtggagc aggctgaggctctgaaggcc aagggagtcc aggtggtggc 480 ctgtctgagt gttaatgatg cctttttgactggcgagtgg ggccgagccc acaaggcgga 540 aggcaaggtt cggctcctgg ctgatcccactggggccttt gggaaggaga cagacttatt 600 actagatgat tcgctggtgt ccatctttgggaatcgangt ctcaagaggt tctccatggt 660 ggtanaggat ggcatagtga aggccctgaatgtggaacca gatggcanag gcctcanctg 720 cagcctggna cccaatatca tctcanagctctgaggccct gggacagatt acttcttcan 780 ccctccctat ntcanctgcc cagccctgtgctggggccct gcaattggaa tgttgggcag 840 a 841 4 253 DNA Homo sapiensmisc_feature Incyte ID No 743725H1 4 ggcggcccag gcccgtcttn cgcaggtgtcgccgctgtgc cgctagcggt gccccgnctg 60 ctgcggtggc accagccagg aggcggagtggaagtggccn tggggcgggt atgggactag 120 ctggcgtgtg cgccctgaga cggtnagcgggctatatact cgtcggtggg gccngcggtc 180 antctgnggc agggcagcaa gacggtgcagtgaaggagag tnggcgtctn gcggggtccc 240 cagtttcagc aga 253 5 248 DNA Homosapiens misc_feature Incyte ID No 2521256H1 5 ggcggcccag gcccgccttccgcagggtgt cgccgctgtg ccgctagcng tgccccgcct 60 gctgcggtgg caccagccaggaggcggagt ggaagtggcc gtggggcggg tatgggacta 120 gctggcgtgt gcgccctgagacgctcagcg ggctatatac tcgtcggtgg ggccggcggt 180 nagtctgcgg cagggcagcaagacggtgca gtgaaggaga gtgggcgtct ggcgnggtcc 240 gcagtttc 248 6 252 DNAHomo sapiens misc_feature Incyte ID No 602137H1 6 gtcgccgctg tgccgctagcggtgccccgc ctgctgcggt ggcaccagcc agggaggcgg 60 agtggaagtg gccgtggggcgggtatggga ctagctggcg tgtgcgccct gagacgnttc 120 agcggggctt atatacttcgttcgggtggg ggcccgggcg ggttcaagtc ntgcgggcca 180 accgggcaag ccaaaggaccgggttgccaa gttgaaaagg gaggaagttt gggncngttc 240 ttttgncggg gg 252 7 249DNA Homo sapiens misc_feature Incyte ID No 2373064H1 7 tgcagccatggccccaatca aggtgggaga tgccatccca gcagtggagg tgtttgaagg 60 ggagccagggaacaaggtga acctggcaga gctgttcaag ggcaagaagg gtgtgctgtt 120 tggagttcctggggccttca cccctggatg ttccaagaca cacctgccag ggtttgtgga 180 gcaggctgaggctctgaagg ccaagggagt ccaggtggtg gcctgtctga gtgttaatga 240 tgcctttgt 2498 138 DNA Homo sapiens misc_feature Incyte ID No 911226H1 8 ctggggcctttgggaaggag acagacttat tactagatga ttcgntggtg tccatctttg 60 ggaatcgacgtntcaagagg ttctncatgg tggtacagga tggcatagtg aaggccctga 120 atgtggaaccagatggca 138 9 182 DNA Homo sapiens misc_feature Incyte ID No 2226546H19 accagatggc acaggcctca cctgcagcct ggcacccaat atcatctcac agctctgagg 60ccctgggcca gattacttcc tccacccctc cctatctcac ctgcccagcc gtgtgctggg 120gccctgcaat tggaatgttg gccagatttc tgcaataaac acttgtggtt tgcggccaaa 180 aa182 10 504 DNA Homo sapiens misc_feature Incyte ID No 1732084X15 10aaggtgaacc tggcagagtg ttcaagggca agaagggtgt gctgtttgga gttcctgggg 60ccttcacccc tggatgttcc aagacacacc tgccagggtt tgtggagcag gctgaggctc 120tgaaggccaa gggagtccag gtggtggcct gtctgagtgt taatgatgcc tttgtgactg 180gcgagtgggg ccgagcccac aaggcggaag gcaaggttcg gctcctggct gatcccactg 240ggnggncttg gaaggagcag acttattact agatgattcg ctggtgtcca tctttgggga 300atcgacgtct caagaggttc tccatggtgg tnacaggatg gcatagtgaa ggccctgaat 360gtggaaccag atggcacagg nctcaactgn agccttgcac ccaatatcat ctcacagctc 420tgaggnnctn gnccagatta cttnctcaat ntccctctaa cgcatntgac cagccctgtg 480gcatgggccc nnnaaatggn atnt 504 11 261 DNA Macca fascicularismisc_feature Incyte ID No 700459782H1 11 cgacagcagc agcaagacggcgaagtgaag gagggtgggc gtctggcggg gtccgcagtt 60 tcagcagaac cgctgcagccatggccccga tcaaggtggg agatgccatc cctgcagtgg 120 aggtgtttga aggggagccagggaacaagg tgaacctggc agagctgttc aagggcaaga 180 agggtgtgct gtttggagttcccggggcct tcacgcctgg atgttccaga cccacctacc 240 agggtttgtg gagcaggctg a261 12 270 DNA Macca fascicularis misc_feature Incyte ID No 700711869H112 gggactagcc ggcgtgtgcg tcctgagacg ctcagcgggc tatatactcg gtggggccgc 60cggtcagtct gtggcagcga cagcagcagc aagacggcga agtgacggag ggtgggcgtc 120tggcggggtc cgcagtttca gcagaaccgc tgcagccatg gccccgatca aggtgggaga 180tgccatccct gcagtggagg tgtttgaagg ggagccaggg aacaaggtga acctggcaga 240gctgttcaag ggcaagaagg gtgtgctgtt 270 13 246 DNA Macca fascicularismisc_feature Incyte ID No 700711005H2 13 ggcgggtatg ggactagccggcgtgtgcgt cctgagacgc tcagcgggct atatactcgg 60 tggggccgcc ggtcagtctgtggcagcgac agcagcagca agacggcgaa gtgaaggagg 120 gtgggcgtct ggcggggtccgcagtttcag cagaaccgct gcagccatgg ccccgatcaa 180 ggtgggagat gccatccctgcagtggaggt gtttgaaggg gagccaggga acaaggtgaa 240 cctggc 246 14 246 DNAMacca fascicularis misc_feature Incyte ID No 700712820H1 14 gcggagtgaaagtggtctta gggcgggtat gggactagcc ggcgtgtgcg tcctgagacg 60 ctcagcgggctatatactcg gtggggccgc cggtcagtct gtggcagcga cagcagcagc 120 aagacggcgaagtgaaggag ggtgggcgtc tggcggggtc cgcagtttca gcagaaccgc 180 tgcagccatggccccgatca aggtgggaga tgccatccct gcagtggagg tgtttgaagg 240 ggagcc 246 15254 DNA Macca fascicularis misc_feature Incyte ID No 700718769H1 15cccagctagg gggcggagtg aaagtggtct tagggcgggt atgggactag ccggcgtgtg 60cgtcctgaga cgctcagcgg gctatatact cggtggggcc gccggtcagt ctgtggcagc 120gacagcagca gcaagacggc gaagtgaagg agggtgggcg tctgcggggt ccgcagtttc 180agcagaaccg ctgcagccat ggccccgatc aaggtgggag atgccatccc tgcagtggag 240gtgtttgaag ggga 254 16 152 DNA Macca fascicularis misc_feature Incyte IDNo 700715209H1 16 cattcgttag tgtccatctt tgggaatcga cgtctcaaga ggttctccatggtggtacag 60 gatggcatag tgaaggccct gaatgtggaa ccagatggca caggcctcacctgcagtctg 120 gcacccagca tcatctcaca gctctgaggc cc 152 17 134 DNA Maccafascicularis misc_feature Incyte ID No 700708715H1 17 gcgacacagcagcaagacgg cgaagtgaag gagggtgggc gtctggcggg gtccgcagtt 60 tcagcagaaccgctgcagcc atggccccga tcaaggtggg agatgccatc cctgcagtgg 120 aggtgttgaagggg 134 18 388 DNA Macca fascicularis misc_feature Incyte ID No701738592T1 18 gagatgatgc tgggtgccat actgcatgtt aggcctgtac ccatctggttccacattcag 60 gtccttcact atgccatcct gttccacctt ggagaacctc ttgagcacgtcgattcccaa 120 atgatagagc atctaacgat atcatctagt gatacgtctg tgtaccttcccataatggtc 180 ccggtgcgga tctgccatga gtcggaacct gcagagaaag gaaaggtcacaagaagaata 240 tcctacctgg ccctgactcc ctgcaggccc cacacctcac cttgccttccgccttgtggg 300 ctcggcccca ctcgccagtt acaaagtcat cattaacact cagacaggccaacacctgga 360 ctcccttggc cttcagagcc tcagcctg 388 19 231 DNA Maccafascicularis misc_feature Incyte ID No 700459993H2 19 cagcagtgcctccagccgtg cgccgcctgc tgcggtggac ccagctaggg ggcggagtga 60 aagtggtcttagggcgggta tgggactagc cggcgtgtgc gtcctgagac gctcagcggg 120 ctatatactcggtggggacg ccggtcagtc tgtggcagcg acagagcagc aagacggcga 180 agtgaaggagggtgggcgtc tagcggggtc cgcagtttca gcagaaccgc t 231 20 266 DNA Musmusculus misc_feature Incyte ID No 701087440H1 20 cgtgcatcga cgtgcttggcaggcagagca ggccgcaaag aagcaggttg ggagtgtggc 60 ggagacgcag cttcagcagctccgcggtga ccatggcccc gatcaaggtg ggagatgcca 120 ttccctcagt ggaggtatttgaaggggaac cgggaaagaa ggtgaacttg gcagagctgt 180 tcaagggcaa gaaaggtgttttgtttggag tccctggggc atttacacct ggctgttcta 240 agacccacct gcctgggtttgtggag 266 21 273 DNA Mus musculus misc_feature Incyte ID No 701253435H121 cgagtcctgg gctgcaaagc cagttctgtg ctccgtgcat cgacgtgctt ggcaggcaga 60gcaggccgga aagaagcagg ttgggagtgt ggcggagccg cagcttcagc agctccgcgg 120tgaccatggc cccgatcaag gtgggagatg ccattccctc agtggaggta tttgaagggg 180aaccgggaaa gaaggtgaac ttggcagagc tgttcaaggg caagaaaggt gttttgtttg 240gagtccctgg ggcatttaca cctggctgtt cta 273 22 231 DNA Mus musculusmisc_feature Incyte ID No 701424530H1 22 ctgggctgca aagccagttctgtgctccgt gcatcgacgt gcttggcagg cagagcaggc 60 cggaaagaag caggttgggagtgtggcgga gcccgcagct tcagcagctc cgcggtgacc 120 atggccccga tcaaggtgggagatgccatt ccctcagtgg aggtatttga aggggaaccg 180 ggaaagaagg tgaacttggcagagctgttc aagggcaaga aaggtgtttt g 231 23 248 DNA Mus musculusmisc_feature Incyte ID No 701423726H1 23 gccatctgcg ggtgggctgcggcagcgggt cggctatgct acagctgggg cttcgagtcc 60 tgggctgcaa agccagttctgtgctccgtg catcgacgtg cttggcaggc agagcaggcc 120 ggaaagaagc aggctgggagtgtggcggag cccgcagctt cagcagctcc gcggtgacca 180 tggccccgat caaggtgggagatgccattc cctcagtgga ggtatttgaa ggggaaccgg 240 gaaagaag 248 24 560 DNARattus norvegicus misc_feature Incyte ID No 702154193H1 24 gccagaggcaaagacacaaa ttaaactgtt tattgcagaa atattgtcaa ggttccattc 60 cagtggccctcaggtaagga ggtactgggc ctttggccca gagctgggtg gaggagatgg 120 gagagtcagaggacattctg gtcagggcct cagagttgtg agaggatgtt gggggccagg 180 ctgcaggtgaggcctgtgcc atccggctcc acgttcagtg cctttactac acccttgtct 240 atcaccatggagaacctttt tagccgacga ttcccaaaga gagacaccaa agaatcatct 300 agtagtaaatctgtctcctt tccaaaagct ccagtggggt cagccaggag ctgaaccttg 360 ccttctgcctggtgggctcg accccactct gcagtcacga agacatcatt aacactcaga 420 caggccaccacttgtgctcc cttggccttc agagctccgg cttgctccac aaacccaggc 480 agatgggtcttggaacagcc aggtgtaaat gccccaggga ctccaaacaa aacacctttc 540 ttgtccttgaacagctctgc 560 25 464 DNA Rattus norvegicus misc_feature Incyte ID No702242583H1 25 ggcagagtca tctgcgggtg ggctgcggcg gcggggcggg catggtccagctgaggtttt 60 gcgtcctagg cagcatagcc ggatcggtgc tccgtgcatc ggctacttggacgtgcgtgg 120 caggcagagc aggccggaaa ggagcaggtt gggagtgtgg tggggccnctgcagcttaca 180 gcagtgccgc ggtgactatt ggcccctgat caaggtggga gacaccattccctcagtgga 240 ggtatttgaa ggggaacctg gaaagaaggt gaacttggca gagctgttcaaggacaagaa 300 aggtgttttg tttggagtcc ctggggcatt tacacctggc tgttccaagacccatctgcc 360 tgggtttgtg gagcaagccg gagctctgaa ggccaaggga gcacaagtggtggcctgtct 420 gagtgttaat gatgtcttcg tgactgcaga gtggggtcga gccc 464 262805 DNA Homo sapiens misc_feature Incyte ID No 2822412CB1 26 gttgcggtccgcttcggttt ctgttgcggg acccggggtg tctcctagcg caaccggaac 60 tagccttctgggggccggct tcctttatct ctggcggcct tgtagtcgtc tccgagactc 120 cccacccctccttccctctt gaccccctag gtttgattgc cctttccccg aaacaactat 180 catgagcgcgaggctgccgg tgttgtctcc acctcggtgg ccgcggctgt tgctgctgtc 240 gctgctcctgctgggggcgg ttcctggccc gcgccggagc ggcgctttct acctgcccgg 300 cctggcgcccgtcaacttct gcgacgaaga aaaaaagagc gacgagtgca aggccgaaat 360 agaactatttgtgaacagac ttgattcagt ggaatcagtt cttccttatg aatacacagc 420 gtttgatttttgccaagcat cagaaggaaa gcgcccatct gaaaatcttg gtcaggtact 480 attcggggaaagaattgaac cttcaccata taagtttacg tttaataaga aggagacctg 540 taagcttgtttgtacaaaaa cataccatac agagaaagct gaagacaaac aaaagttaga 600 attcttgaaaaaaagcatgt tattgaatta tcaacatcac tggattgtgg ataatatgcc 660 tgtaacgtggtgttacgatg ttgaagatgg tcagaggttc tgtaatcctg gatttcctat 720 tggctgttacattacagata aaggccatgc aaaagatgcc tgtgttatta gttcagattt 780 ccatgaaagagatacatttt acatcttcaa ccatgttgac atcaaaatat actatcatgt 840 tgttgaaactgggtccatgg gagcaagatt agtggctgct aaacttgaac cgaaaagctt 900 caaacatacccatatagata aaccagactg ctcagggccc cccatggaca taagtaacaa 960 ggcttctggggagataaaaa ttgcctatac ttactctgtt agcttcgagg aagatgataa 1020 gatcagatgggcgtctagat gggactatat tctggagtct atgcctcata cccacattca 1080 gtggtttagcattatgaatt ccctggtcat tgttctcttc ttatctggaa tggtagctat 1140 gattatgttacggacactgc acaaagatat tgctagatat aatcagatgg actctacgga 1200 agatgcccaggaagaatttg gctggaaact tgttcatggt gatatattcc gtcctccaag 1260 aaaagggatgctgctatcag tctttctagg atccgggaca cagattttaa ttatgacctt 1320 tgtgactctatttttcgctt gcctgggatt tttgtcacct gccaaccgag gagcgctgat 1380 gacgtgtgctgtggtcctgt gggtgctgct gggcacccct gcaggctatg ttgctgccag 1440 attctataagtcctttggag gtgagaagtg gaaaacaaat gttttattaa catcatttct 1500 ttgtcctgggattgtatttg ctgacttctt tataatgaat ctgatcctct ggggagaagg 1560 atcttcagcagctattcctt ttgggacact ggttgccata ttggcccttt ggttctgcat 1620 atctgtgcctctgacgttta ttggtgcata ctttggtttt aagaagaatg ccattgaaca 1680 cccagttcgaaccaatcaga ttccacgtca gattcctgaa cagtcgttct acacgaagcc 1740 cttgcctggtattatcatgg gagggatttt gccctttggc tgcatcttta tacaactttt 1800 cttcattctgaatagtattt ggtcacacca gatgtattac atgtttggct tcctatttct 1860 ggtgtttatcattttggtta ttacctgttc tgaagcaact atacttcttt gctatttcca 1920 cctatgtgcagaggattatc attggcaatg gcgttcattc cttacgagtg gctttactgc 1980 agtttatttcttaatctatg cagtacacta cttcttttca aaactgcaga tcacgggaac 2040 agcaagcacaattctgtact ttggttatac catgataatg gttttgatct tctttctttt 2100 tacaggaacaattggcttct ttgcatgctt ttggtttgtt accaaaatat acagtgtggt 2160 gaaggttgactgaagaagtc cagtgtgtcc agttaaaaca gaaataaatt aaactcttca 2220 tcaacaaagacctgtttttg tgactgcctt gagttttatc agaattattg gcctagtaat 2280 ccttcagaaacaccgtaatt ctaaataaac ctcttcccat acacctttcc cccataagat 2340 gtgtcttcaacactataaag catttgtatt gtgatttgat taagtatata tttggttgtt 2400 ctcaatgaagagcaaattta aatattatgt gcatttgtaa atacagtagc tataaaattt 2460 tccatacttctaatggcaga atagaggagg ccatattaaa taatactgat gaaaggcagg 2520 acactgcattgtaaatagga ttttctaggc tcggtaggca gaaagaatta tttttctttg 2580 aaggaaataactttttatca tggtaatttt gaaggatgat tcctatgatg tgttcaccag 2640 gggaatgtggcttttaaaga aaatcttcta ttggttgtaa ctgttcatat cttcttactt 2700 ttctgtgttgacttcattat tcccatggta ttggcctttt aaactatgtg cctctgagtc 2760 tttcaatttataaatttgtt atcttaataa atattataaa aatga 2805 27 288 DNA Homo sapiensmisc_feature Incyte ID No 2822412H1 27 atctggaatg gtagctatga ttatgttacggacactgcac aaagatattg ctagatataa 60 tcagatggac tctacggaag atgcccaggaagaatttggc tggaaacttg ttcatggtga 120 tatattccgt cctccaagaa aagggatgctgctatcagtc tttctaggat ccgggacaca 180 gattttaatt atgacctttg tgactctatttttcgctgcc tgggattttg tcacctgcca 240 accgaggagc gctgatgacg tgtgctgtggtcctgtgggt gctgctgg 288 28 234 DNA Homo sapiens misc_feature Incyte IDNo 3236331H1 28 gttgcggtcc gcttcggttt ctgttgcggg acccggggtg tctcctagcgcaaccggaac 60 tagccttctg ggggccggct tcctttatct ctggcggcct tgtagtcgtctccgagattn 120 ccccanccct ccttccctct tganccccta ggtttgattg ccctttccccgaaacaacta 180 tcatgagcgc gaggctgccg gtgttgtctc cacctcggtg gccgcggctgttgc 234 29 579 DNA Homo sapiens misc_feature Incyte ID No 269777R1 29ccgagactnc ccacccctcc ttccctcttg accccctagg tttgattgcc ctttccccga 60aacaactatc atgagcgcga ggctgccggt gttgtctcca cctcggtggc cgcggctgtt 120gctgctgtcg ctgctcctgc tgggggcggt tcctggcccg cgccggagcg gcgctttcta 180cctgcccggc ctggcgcccg tcaacttctg cgacgaagaa aaaaagagcg acgagtgcaa 240ggccgaaata gaactatttg tgaacagact tgattcagtg gaatcagttc ttccttatga 300atacacagcg tttgattttt gccaagcatc agaaggaaag cgcccatctg aaaatcttgg 360tcaggtacta ttcggggaaa gaattgaacc ttcaccatat aagtttacgt ttaataagaa 420ggagacctgt aagcttgttt gtacaaaaac ataccataca gagaaagctg aagacaaaca 480aaagttagaa ttcttgaaaa aaagcatgtt attgaattat caacatcact ggattgtgga 540taatatgcct gtaacggtgg tgttacggat gttgaagat 579 30 529 DNA Homo sapiensmisc_feature Incyte ID No 1359919F1 30 ttttgccaag catcagaagg aaagcgcccatctgaaaatc ttggtcaggt actattcggg 60 gaaagaattg aaccttcacc atataagtttacgtttaata agaaggagac ctgtaagctt 120 gtttgtacaa aaacatacca tacagagaaagctgaagaca aacaaaagtt agaattcttg 180 aaaaaaagca tgttattgaa ttatcaacatcactggattg tggataatat gcctgtaacg 240 tggtgttacg atgttgaaga tggtcagaggttctgtaatc ctggatttcc tattggctgt 300 tacattacag ataaaggccn tgcaaaagatgcctgtgtta ttagttcaga tttccatgaa 360 agagatacat tttacatctt caaccatgttgacatcaaaa tatactatca tgttgttgaa 420 actgggtcca tgggagcnag attagtggctgctaacttga accgaaaagc ttcaaacata 480 cccatatagg ataaaccaga ctgctcagggccccccatgg ccttaagta 529 31 492 DNA Homo sapiens misc_feature Incyte IDNo 770535R1 31 tccaagaaaa gggatgctgc tatcagtctt tctaggatcc gggacacagattttaattat 60 gacctttgtg actctatttt tcgcttgcct gggatttttg tcacctgccaaccgaggagc 120 gctgatgacg tgtgctgtgg tcctgtgggt gctgctgggc acccctgcaggctatgttgc 180 tgccagattc tataagtcct ttggaggtga gaagtggaaa acaaatgttttattaacatc 240 atttctttgt cctgggattg tatttgctga cttctttata atgaatctgatcctctgggg 300 agaaggatct tcagcagcta ttccttttgg gacactggtt gccatattggccctttggtt 360 ctgcatatct gtgcctctga cgtttattgg tgcatacttt ggttttaagaagaatgccat 420 tgaacaccca gttcgaacca atcagattcc acgtcagatt cctgaacagtcgttctacac 480 gaagcccttg cc 492 32 458 DNA Homo sapiens misc_featureIncyte ID No 002505H1 32 aaggatcttc agcagctatt ccttttggga cactggttgccatattggcc ctttggttct 60 gcatatctgt gcctctgacg tttattggtg catactttggttttaagaag aatgccattg 120 aacacccagt tcgnaccaat cagattccac gtcagattcctgaacagtcg ttctacacga 180 agcccttgcc tggtattatc atgggaggga ttttgccctttggctgcatc ttttatacaa 240 ctttnnttca ttctgaatag tatttggtca caccagatgtattacatgtt tggcttccta 300 tttctggtgt ttatcanttt gggttattna cctgttctgaagcaactata cttcctttgc 360 tatttccacc tatgttcaga gggtttncaa ttggaaatggggtcantcct tccgggtggn 420 ttnctcaagt ttnttctaac ccntcgagac actncggg 45833 300 DNA Homo sapiens misc_feature Incyte ID No 896216H1 33 ctttatacaacttttcttca ttctgaatag tatttggtca caccagatgt attacatgtt 60 tggcttcctatttctggtgt ttatcatttt ggttattacc tgttctgaag caactatact 120 tctttgctattttccaccta tgtgcagagg attatcattg gcaatggcgt tcattcctta 180 cgagtggctttactgcagtt tatttcttaa tctatgcagt acactacttc tnttcaaaac 240 tgcagatcacgggaacagca agcacaattc tgtactttgg ttataccatg ataatggttt 300 34 182 DNAHomo sapiens misc_feature Incyte ID No 741936H1 34 ataccatgat aatggttttgatcttctttc tttttacagg aacaattggn ttctttgcat 60 gcttttggtt tgttaccaaaatatacagtg tggtgaaggt tgactgaaga agtncagtgt 120 gtncagttaa aacaggaataanttaaactc ttcatcaaaa aaanaaannn nnggtnngga 180 aa 182 35 260 DNA Homosapiens misc_feature Incyte ID No 2112041H1 35 atggttttga tcttctttctttttacagga acaattggct tctttgcatg cttttggttt 60 gttaccaaaa tatacagtgtggtgaaggtt gactgaagaa gtccagtgtg tccagttaaa 120 acagaaataa attaaactcttcatcaacaa agacctgttt ttgtgactgc cttgagtttt 180 atcagaatta ttggcctagtaatccttcag aaacaccgta attctaaata aacctcttcc 240 catacacctt tcccccataa260 36 391 DNA Homo sapiens misc_feature Incyte ID No 2132059R6 36cagaattatt ggcctagtaa tccttcagaa acaccgtaat tctaaataaa cctcttccca 60tacacctttc ccccataaga tgtgtcttca acactataaa gcatttgtat tgtgatttga 120ttaagtatat atttggttgt tctcaatgaa gagcaaattt aaatattatg tgcatttgta 180aatacagtag ctataaaatt ttccatactt ctaatggcag aatagaggag gccatattaa 240ataatactga tgaaaggcag gacactgcat tgtaaatagg attttctagg ctcggtaggc 300agaaagaatt atttttcttt gaaggaaata actttttatc atggtaattt tgaaggatga 360ttcctatgga tgtgttcacc caggggatgt g 391 37 610 DNA Homo sapiensmisc_feature Incyte ID No 1609872X13 37 ctatacttac tctgttagct tcgaggaagatgataagatc agatgggcgt ctagatggga 60 ctatattctg gagtctatgc ctcatacccacattcagtgg tttagcatta tgaattccct 120 ggtcattgtt ctcttcttat ctggaatggtanctatgatt atgttacgga cactgcacaa 180 agatatngct aganataatc anatggactctacngaagat gcccaggaag aanttggctg 240 gaaacttgtt catggtgata nattccgtcctccatnanaa gggatgctgc tatcantctt 300 tctangatcc gngacacaga ttttaattatgacctttntg actcnatntt ncgcttgcct 360 gagattttng ttcacctgcc aaccnaggagcgcngatgac gtgtgctgtg gtcctnnngg 420 tgctgctggg cacccctgca ggctatgttgctgccagatt cnataagtcc tctggaggtg 480 nnaagtggac aacaaatgtg tnantaacatcattcnttgn cctngattgt nttgngactc 540 atanatgatc nanctctggg agagatctcgcagcatcttt tggancggtg caatnaccct 600 ggtcncaacg 610 38 87 DNA Maccafascicularis misc_feature Incyte ID No 700708848H1 38 gctgatgacgtgtgctgtgg cacagtgggt gctgctgggc acccctggca ggctatgttg 60 ctgccagattctataagtcc tttggag 87 39 294 DNA Mus musculus misc_feature Incyte ID No700108409H2 39 ggccccccat gatgatgcca ggcaaaggct ttgtgtagaa cgactgctcaggaatctgac 60 gagggatctg attggttcga actgggtgtt caatggcatt cttcttaaagccaaagtatg 120 caccaataaa cgtcaaaggc acagatatgc agaaccagag ggccaagatggcaaccagag 180 tgccaaaagg aatggctgct gaagagcctt ctccccagag gattagattcatgataaaga 240 agtcagcaaa cacaatccca ggacaaagaa atgatgtcaa taaaacatttgttt 294 40 288 DNA Mus musculus misc_feature Incyte ID No 701091450H140 atgggagcaa gattagtggc tgctaaactt gaaccaaaaa gcttcaagca tacccacata 60gataaaccag actgctctgg acctgccatg gatataagca acaaggcttc aggagagatc 120aaaattgcct atacttattc tattagtttt gaggaagaga aaaacatcag atgggcgtct 180aggtgggact atattctgga gtctatgcct cacacccata ttcagtggtt tagcataatg 240aactccttgg ttattgtcct cttcttatct ggaatggtag ctatgatt 288 41 252 DNA Musmusculus misc_feature Incyte ID No 701080359H1 41 ccaaagacct gttttgtgactaccttgagt tttctcagac ttattggcct agtaatcctt 60 cagaaacaac atacttccaagtccacctct cccatgcacc tgcacctaca agatgtgttt 120 caacactaaa gcatttgtattgtgattgga ttaagtatat attcagttgt tctcagtgaa 180 gagcagattt aaatattatgtgcatttgta aatacgatag ctataaactt ttcaatactt 240 ctaatggcag at 252 42 435DNA Canis familiaris misc_feature Incyte ID No 702775324H1 42 ctgaatagtatctggtcaca ccagatgtat tacatgtttg gcttcctatt tctggtattt 60 atcattttggttattacatg ttcagaagca actatacttc tttgctattt ccacctatgt 120 gcagaggattatcattggca gtggcgttca ttccttacca gtggctttac tgcagtttat 180 ttcttaatatatgcaataca ctacttcttt tcaaaactgc aaatcacagg aacagcaagt 240 acaattctgtactttggcta taccatgata atggttttga tcttctttct ttttacagga 300 acaattggcttctttgcatg cttttggttt gttaccaaaa tatacagtgt ggtgaaggtt 360 gactgaagaagccagtgtgt ccagttaaaa cagaaataaa ttaaattctt catcaacaaa 420 gagctgtttttgtga 435 43 541 DNA Rattus norvegicus misc_feature Incyte ID No702237054H1 43 cagttcgaac caatcagatt cctcgtcaga ttcccgagca gtcgcgtctacaccaagcct 60 ttgcccggca tcatcatggg gggcattttg cccttcggct gcatctttatacagcttttc 120 ttcattctca acagcattgg gtcacaccag atgtattaca tgtttggcttcctgtttctg 180 gtgtttatca tgttggttat tacatgctcg gaggcaacca tacttctttgctattttcat 240 ctatgtgcag aggattacca ttggcagtgg cgttccttcc tcaccagcggcttcacggca 300 gtgtatttcc tcgtatacgc catacactac ttcttttcaa aactgcagatcacgggaaca 360 gcaagtacaa tcctgtactt tggttacact atgataatgg tcttgatcttcttccttttt 420 acaggaacaa ttggcttctt tgcatgcttt tggttcgtca ccaaaatatacagtgtggtg 480 aaggtcgact gaagaaaccc agtgtgtcca gttaaaacaa ataaactaaatcttcatcca 540 c 541 44 528 DNA Rattus norvegicus misc_feature Incyte IDNo 702212516H1 44 tctatatcat gaatctgatc ctctggggag agggctcttc agcagcgattccttttggca 60 ccctggttgc catcttggcc ctctggttct gcatatctgt gcctttgacgtttattggtg 120 catattttgg ctttaagaag aatgccattg aacacccagt tcgaaccaatcagattcctc 180 gtcagattcc cgagcagtcc ttctacacca agcctttgcc cggcatcatcatggggggca 240 ttttgccctt cggctgcatc tttatacagc ttttcttcat tctcaacagcatttggtcac 300 accagatgta ttacatgttt ggcttcctgt ttctggtgtt tatcattttggttattacat 360 gctcggaggc aaccatactt ctttgctatt ttcatctatg tgcagaggattaccattggc 420 agtggcgttc cttcctcacc agcggcttca cggcagtgta tttcctcgtatacgccatac 480 actacttctt ttcaaaactg cagatcacgg gaacagcaag tacaatcc 52845 574 DNA Rattus norvegicus misc_feature Incyte ID No 702599525T1 45aataatgatg tcaatcacag aaaagtatga agatataaca attacaacca atacaagatt 60tttataagcc acatcccccc aatgaacaca tcataggaat catccttcaa aattaccatg 120ataaaaagtt atttccttca aagaaaaata attctttcca cctaccgagg tctagaaaac 180cctatgtaca atcgactgtc cagtatcgtc agtattatgg cctcctctaa tctgccatta 240gaagtattga aaagtttata gctaccgtat ttacaaatgc acataatatt taaatttgct 300cttcactgag aacaactgaa tatatactta atccaatcac aatacaaatg ctttagtgtt 360gaaatacatc ttgtaggtag ggacaggcgc atggggagag gtgtacttgg aagtctgctg 420tttctgaagg attactaagc cataagtctg agaaaactca agggagtcac aacaacaggt 480ctttgtggat gaagatttag tagattagtt ataactggac acactggggt ttctacagtc 540gagcttcaac cctacgagcc gaaattccga gctt 574 46 293 DNA Rattus norvegicusmisc_feature Incyte ID No 700228705H1 46 acttactcta ttagttttgaggaagagaaa aacatcaggt gggcctccag atgggactac 60 attctggagt ctatgcctcacactcacatt caatggttta gcataatgaa ttccttggtg 120 attgtcctct tcttgtctggaatggtagct atgattatgt tacgcacact acataaagat 180 attgccagat ataaccagatggactctacg gaggatgccc aggaagaatt tggctggaaa 240 ctcgttcatg gggatatattccgtcctcca agaaagggga tgctgctgtc tgt 293 47 287 DNA Rattus norvegicusmisc_feature Incyte ID No 700230086H1 47 ctatttttcg catgtctgggattcttgtcc cctgccaatc gaggacccct gatgacgtgt 60 gctgtggtct tgtgggtgctactgggcaca cctgctggct atgttgctgc cagattctac 120 aagtcctttg ggggcgagaagtggaaaaca aatgttttat tgacatcctt cctttgtcct 180 gggattgtgt ttgctgacttctttatcatg aatctgatcc tctggggaga gggctcttca 240 gcagcgattc cttttggcaccctggttgcc atcttggccc tctggtt 287 48 267 DNA Rattus norvegicusmisc_feature Incyte ID No 700776642H1 48 aagaaagggg atgctgctgtctgtctttct aggatctgga acacagattt taattatgac 60 ttttgtaact ctatttttcgcatgtctggg attcttgtcc cctgccaatc gaggagccct 120 gatgacgtgt gctgtggtcttgtgggtgct actgggcaca cctgctggct atgttgctgc 180 cagattctac aagtcctttgggggcgagaa gtggaaaaca aatgttttat tgacatcctt 240 cctttgtcct gggattgtgtttgctga 267 49 298 DNA Rattus norvegicus misc_feature Incyte ID No700545190H1 49 atcttcttac ttttctgtgt tgacttcatt attcccatgg tattggccttttaaactatg 60 tgcctctgag tctttggatt tataaatttg ttatcttaat aaatattgtaaaaatgcctt 120 cattgtatca ttcccagcat atgaagaaaa ctctgtgaat acagatgtgctgaccactat 180 actgttctat cacatgatgt tgactactgt agacacacac tttctgtctccacctaagtg 240 aaattttagt cttttgggct ttgtcaagtc tgagttttca ctctgaaagaatgctaga 298 50 1980 DNA Rattus norvegicus misc_feature Incyte ID No700228705.con 50 acttactcta ttagttttga ggaagagaaa aacatcaggt gggcctccagatgggactac 60 attctggagt ctatgcctca cactcacatt caatggttta gcataatgaattccttggtg 120 attgtcctct tcttgtctgg aatggtagct atgattatgt tacgcacactacataaagat 180 attgccagat ataaccagat ggactctacg gaggatgccc aggaagaatttggctggaaa 240 ctcgttcatg gggatatatt ccgtcctcca agaaagggga tgctgctgtctgtctttcta 300 ggatctggaa cacagatttt aattatgact tttgtaactc tatttttcgcatgtctggga 360 ttcttgtccc ctgccaatcg aggagccctg atgacgtgtg ctgtggtcttgtgggtgcta 420 ctgggcacac ctgctggcta tgttgctgcc agattctaca agtcctttgggggcgagaag 480 tggaaaacaa atgttttatt gacatccttc ctttgtcctg ggattgtgtttgctgacttc 540 tttatcatga atctgatcct ctggggagag ggctcttcag cagcgattccttttggcacc 600 ctggttgcca tcttggccct ctggttctgc atatctgtgc ctttgacgtttattggtgca 660 tattttggct ttaagaagaa tgccattgaa cacccagttc gaaccaatcagattcctcgt 720 cagattcccg agcagtccgt ctacaccaag cctttgcccg gcatcatcatggggggcatt 780 ttgcccttcg gctgcatctt tatacagctt ttcttcattc tcaacagcatttggtcacac 840 cagatgtatt acatgtttgg cttcctgttt ctggtgttta tcattttggttattacatgc 900 tcggaggcaa ccatacttct ttgctatttt catctatgtg cagaggattaccattggcag 960 tggcgttcct tcctcaccag cggcttcacg gcagtgtatt tcctcgtatacgccatacac 1020 tacttctttt caaaactgca gatcacggga acagcaagta caatcctgtactttggttac 1080 actatgataa tggtcttgat cttcttcctt tttacaggaa caattggcttctttgcatgc 1140 ttttggtttg tcaccaaaat atacagtgtg gtgaaggtcg actgaagaaacccagtgtgt 1200 ccagttaaaa caaataaact aaatcttcat ccacaaagac ctgttttgtgactcccttga 1260 gttttctcag acttatggcc tagtaatcct tcagaaacag cagacttccaagtacacctc 1320 tccccatgcg cctgtcccta cctacaagat gtatttcaac actaaagcatttgtattgtg 1380 attggattaa gtatatattc agttgttctc agtgaagagc aaatttaaatattatgtgca 1440 tttgtaaata cggtagctat aaacttttca atacttctaa tggcagattagaggaggcca 1500 taatactgac gatactggac agtcgattgt acatagggtt ttctagactcggtaggtgga 1560 aagaattatt tttctttgaa ggaaataact ttttatcatg gtaattttgaaggatgattc 1620 ctatgatgtg ttcattgggg ggatgtggct tttaaaaatc ttgtattggttgtaattgtt 1680 catatcttct tacttttctg tgttgacttc attattccca tggtattggccttttaaact 1740 atgtgcctct gagtctttgg atttataaat ttgttatctt aataaatattgtaaaaatgc 1800 cttcattgta tcattcccag catatgaaga aaactctgtg aatacagatgtgcctgacca 1860 ctatactgtt ctatcaacat gaatgttgac tactgtagac acacactttctgtctccacc 1920 taagtgaaat tttagtcttt tgggcgtttg tcaagtctga gttctcactctgaaagaatg 1980 51 394 PRT Rattus norvegicus misc_feature Incyte ID No700228705 51 Thr Tyr Ser Ile Ser Phe Glu Glu Glu Lys Asn Ile Arg Trp Ala1 5 10 15 Ser Arg Trp Asp Tyr Ile Leu Glu Ser Met Pro His Thr His Ile 2025 30 Gln Trp Phe Ser Ile Met Asn Ser Leu Val Ile Val Leu Phe Leu 35 4045 Ser Gly Met Val Ala Met Ile Met Leu Arg Thr Leu His Lys Asp 50 55 60Ile Ala Arg Tyr Asn Gln Met Asp Ser Thr Glu Asp Ala Gln Glu 65 70 75 GluPhe Gly Trp Lys Leu Val His Gly Asp Ile Phe Arg Pro Pro 80 85 90 Arg LysGly Met Leu Leu Ser Val Phe Leu Gly Ser Gly Thr Gln 95 100 105 Ile LeuIle Met Thr Phe Val Thr Leu Phe Phe Ala Cys Leu Gly 110 115 120 Phe LeuSer Pro Ala Asn Arg Gly Ala Leu Met Thr Cys Ala Val 125 130 135 Val LeuTrp Val Leu Leu Gly Thr Pro Ala Gly Tyr Val Ala Ala 140 145 150 Arg PheTyr Lys Ser Phe Gly Gly Glu Lys Trp Lys Thr Asn Val 155 160 165 Leu LeuThr Ser Phe Leu Cys Pro Gly Ile Val Phe Ala Asp Phe 170 175 180 Phe IleMet Asn Leu Ile Leu Trp Gly Glu Gly Ser Ser Ala Ala 185 190 195 Ile ProPhe Gly Thr Leu Val Ala Ile Leu Ala Leu Trp Phe Cys 200 205 210 Ile SerVal Pro Leu Thr Phe Ile Gly Ala Tyr Phe Gly Phe Lys 215 220 225 Lys AsnAla Ile Glu His Pro Val Arg Thr Asn Gln Ile Pro Arg 230 235 240 Gln IlePro Glu Gln Ser Val Tyr Thr Lys Pro Leu Pro Gly Ile 245 250 255 Ile MetGly Gly Ile Leu Pro Phe Gly Cys Ile Phe Ile Gln Leu 260 265 270 Phe PheIle Leu Asn Ser Ile Trp Ser His Gln Met Tyr Tyr Met 275 280 285 Phe GlyPhe Leu Phe Leu Val Phe Ile Ile Leu Val Ile Thr Cys 290 295 300 Ser GluAla Thr Ile Leu Leu Cys Tyr Phe His Leu Cys Ala Glu 305 310 315 Asp TyrHis Trp Gln Trp Arg Ser Phe Leu Thr Ser Gly Phe Thr 320 325 330 Ala ValTyr Phe Leu Val Tyr Ala Ile His Tyr Phe Phe Ser Lys 335 340 345 Leu GlnIle Thr Gly Thr Ala Ser Thr Ile Leu Tyr Phe Gly Tyr 350 355 360 Thr MetIle Met Val Leu Ile Phe Phe Leu Phe Thr Gly Thr Ile 365 370 375 Gly PhePhe Ala Cys Phe Trp Phe Val Thr Lys Ile Tyr Ser Val 380 385 390 Val LysVal Asp 52 167 PRT Candida boidinii misc_feature Incyte ID No g170899 52Met Ala Pro Ile Lys Arg Gly Asp Arg Phe Pro Thr Thr Asp Asp 1 5 10 15Val Tyr Tyr Ile Pro Pro Glu Gly Gly Glu Pro Gly Pro Leu Glu 20 25 30 LeuSer Lys Phe Val Lys Thr Lys Lys Phe Val Val Val Ser Val 35 40 45 Pro GlyAla Phe Thr Pro Pro Cys Thr Glu Gln His Leu Pro Gly 50 55 60 Tyr Ile LysAsn Leu Pro Arg Ile Leu Ser Lys Gly Val Asp Phe 65 70 75 Val Leu Val IleSer Gln Asn Asp Pro Phe Val Leu Lys Gly Trp 80 85 90 Lys Lys Glu Leu GlyAla Ala Asp Ala Lys Lys Leu Val Phe Val 95 100 105 Ser Asp Pro Asn LeuLys Leu Thr Lys Lys Leu Gly Ser Thr Ile 110 115 120 Asp Leu Ser Ala IleGly Leu Gly Thr Arg Ser Gly Arg Leu Ala 125 130 135 Leu Ile Val Asn ArgSer Gly Ile Val Glu Tyr Ala Ala Ile Glu 140 145 150 Asn Gly Gly Glu ValAsp Val Ser Thr Ala Gln Lys Ile Ile Ala 155 160 165 Lys Leu 53 189 PRTSynechocystis sp. misc_feature Incyte ID No g1652858 53 Met Thr Pro GluArg Val Pro Ser Val Val Phe Lys Thr Arg Val 1 5 10 15 Arg Asp Glu SerVal Pro Gly Pro Asn Pro Tyr Arg Trp Glu Asp 20 25 30 Lys Thr Thr Glu GlnIle Phe Gly Gly Lys Lys Val Val Leu Phe 35 40 45 Ser Leu Pro Gly Ala PheThr Pro Thr Cys Ser Ser Asn His Leu 50 55 60 Pro Arg Tyr Glu Gln Leu PheGlu Glu Phe Gln Ala Leu Gly Val 65 70 75 Asp Asp Ile Ile Cys Leu Ser ValAsn Asp Ala Phe Val Met Phe 80 85 90 Gln Trp Gly Lys Gln Ile Gly Ala AspLys Val Lys Leu Leu Pro 95 100 105 Asp Gly Asn Gly Glu Phe Thr Arg LysMet Gly Met Leu Val Glu 110 115 120 Lys Ser Asn Leu Gly Phe Gly Met ArgSer Trp Arg Tyr Ser Met 125 130 135 Phe Val Asn Asp Gly Lys Ile Glu LysMet Phe Ile Glu Pro Glu 140 145 150 Phe Gly Asp Asn Cys Pro Val Asp ProPhe Glu Cys Ser Asp Ala 155 160 165 Asp Thr Met Leu Ala Tyr Leu Lys GlyAla Glu Ala Pro Gly Val 170 175 180 Ser Glu Pro Val Lys Ala Phe Val Gly185 54 625 PRT Homo sapiens misc_feature Incyte ID No g1665777 54 MetCys Glu Thr Ser Ala Phe Tyr Val Pro Gly Val Ala Pro Ile 1 5 10 15 AsnPhe His Gln Asn Asp Pro Val Glu Ile Lys Ala Val Lys Leu 20 25 30 Thr SerSer Arg Thr Gln Leu Pro Tyr Glu Tyr Tyr Ser Leu Pro 35 40 45 Phe Cys GlnPro Ser Lys Ile Thr Tyr Lys Ala Glu Asn Leu Gly 50 55 60 Glu Val Leu ArgGly Asp Arg Ile Val Asn Thr Pro Phe Gln Val 65 70 75 Leu Met Asn Ser GluLys Lys Cys Glu Val Leu Cys Ser Gln Ser 80 85 90 Asn Lys Pro Val Thr LeuThr Val Glu Gln Ser Arg Leu Val Ala 95 100 105 Glu Arg Ile Thr Glu AspTyr Tyr Val His Leu Ile Ala Asp Asn 110 115 120 Leu Pro Val Ala Thr ArgLeu Glu Leu Tyr Ser Asn Arg Asp Ser 125 130 135 Asp Asp Lys Lys Lys GluLys Asp Val Gln Phe Glu His Gly Tyr 140 145 150 Arg Leu Gly Phe Thr AspVal Asn Lys Ile Tyr Leu His Asn His 155 160 165 Leu Ser Phe Ile Leu TyrTyr His Arg Glu Asp Met Glu Glu Asp 170 175 180 Gln Glu His Thr Tyr ArgVal Val Arg Phe Glu Val Ile Pro Gln 185 190 195 Ser Ile Arg Leu Glu AspLeu Lys Ala Asp Glu Lys Ser Ser Cys 200 205 210 Thr Leu Pro Glu Gly ThrAsn Ser Ser Pro Gln Glu Ile Asp Pro 215 220 225 Thr Lys Glu Asn Gln LeuTyr Phe Thr Tyr Ser Val His Trp Glu 230 235 240 Glu Ser Asp Ile Lys TrpAla Ser Arg Trp Asp Thr Tyr Leu Thr 245 250 255 Met Ser Asp Val Gln IleHis Trp Phe Ser Ile Ile Asn Ser Val 260 265 270 Val Val Val Phe Phe LeuSer Gly Ile Leu Ser Met Ile Ile Ile 275 280 285 Arg Thr Leu Arg Lys AspIle Ala Asn Tyr Asn Lys Glu Asp Asp 290 295 300 Ile Glu Asp Thr Met GluGlu Ser Gly Trp Lys Leu Val His Gly 305 310 315 Asp Val Phe Arg Pro ProGln Tyr Pro Met Ile Leu Ser Ser Leu 320 325 330 Leu Gly Ser Gly Ile GlnLeu Phe Cys Met Ile Leu Ile Val Ile 335 340 345 Phe Val Ala Met Leu GlyMet Leu Ser Pro Ser Ser Arg Gly Ala 350 355 360 Leu Met Thr Thr Ala CysPhe Leu Phe Met Phe Met Gly Val Phe 365 370 375 Gly Gly Phe Ser Ala GlyArg Leu Tyr Arg Thr Leu Lys Gly His 380 385 390 Arg Trp Lys Lys Gly AlaPhe Cys Thr Ala Thr Leu Tyr Pro Gly 395 400 405 Val Val Phe Gly Ile CysPhe Val Leu Asn Cys Phe Ile Trp Gly 410 415 420 Lys His Ser Ser Gly AlaVal Pro Phe Pro Thr Met Val Ala Leu 425 430 435 Leu Cys Met Trp Phe GlyIle Ser Leu Pro Leu Val Tyr Leu Gly 440 445 450 Tyr Tyr Phe Gly Phe ArgLys Gln Pro Tyr Asp Asn Pro Val Arg 455 460 465 Thr Asn Gln Ile Pro ArgGln Ile Pro Glu Gln Arg Trp Tyr Met 470 475 480 Asn Arg Phe Val Gly IleLeu Met Ala Gly Ile Leu Pro Phe Gly 485 490 495 Ala Met Phe Ile Glu LeuPhe Phe Ile Phe Ser Ala Ile Trp Glu 500 505 510 Asn Gln Phe Tyr Tyr LeuPhe Gly Phe Leu Phe Leu Val Phe Ile 515 520 525 Ile Leu Val Val Ser CysSer Gln Ile Ser Ile Val Met Val Tyr 530 535 540 Phe Gln Leu Cys Ala GluAsp Tyr Arg Trp Trp Trp Arg Asn Phe 545 550 555 Leu Val Ser Gly Gly SerAla Phe Tyr Val Leu Val Tyr Ala Ile 560 565 570 Phe Tyr Phe Val Asn LysLeu Asp Ile Val Glu Phe Ile Pro Ser 575 580 585 Leu Leu Tyr Phe Gly TyrThr Ala Leu Met Val Leu Ser Phe Trp 590 595 600 Leu Leu Thr Gly Thr IleGly Phe Tyr Ala Ala Tyr Met Phe Val 605 610 615 Arg Lys Ile Tyr Ala AlaVal Lys Ile Asp 620 625 55 667 PRT Saccharomyces cerivisiae misc_featureIncyte ID No g2131246 55 Met Ile Tyr Lys Met Ala His Val Gln Leu Leu LeuLeu Tyr Phe 1 5 10 15 Phe Val Ser Thr Val Lys Ala Phe Tyr Leu Pro GlyVal Ala Pro 20 25 30 Thr Thr Tyr Arg Glu Asn Asp Asn Ile Pro Leu Leu ValAsn His 35 40 45 Leu Thr Pro Ser Met Asn Tyr Gln His Lys Asp Glu Asp GlyAsn 50 55 60 Asn Val Ser Gly Asp Lys Glu Asn Phe Leu Tyr Ser Tyr Asp Tyr65 70 75 Tyr Tyr Asn Arg Phe His Phe Cys Gln Pro Glu Lys Val Glu Lys 8085 90 Gln Pro Glu Ser Leu Gly Ser Val Ile Phe Gly Asp Arg Ile Tyr 95 100105 Asn Ser Pro Phe Gln Leu Asn Met Leu Gln Glu Lys Glu Cys Glu 110 115120 Ser Leu Cys Lys Thr Val Ile Pro Gly Asp Asp Ala Lys Phe Ile 125 130135 Asn Lys Leu Ile Lys Asn Gly Phe Phe Gln Asn Trp Leu Ile Asp 140 145150 Gly Leu Pro Ala Ala Arg Glu Val Tyr Asp Gly Arg Thr Lys Thr 155 160165 Ser Phe Tyr Gly Ala Gly Phe Asn Leu Gly Phe Val Gln Val Thr 170 175180 Gln Gly Thr Asp Ile Glu Ala Thr Pro Lys Gly Ala Glu Thr Thr 185 190195 Asp Lys Asp Val Glu Leu Glu Thr Arg Asn Asp Arg Asn Met Val 200 205210 Lys Thr Tyr Glu Leu Pro Tyr Phe Ala Asn His Phe Asp Ile Met 215 220225 Ile Glu Tyr His Asp Arg Gly Glu Gly Asn Tyr Arg Val Val Gly 230 235240 Val Ile Val Glu Pro Val Ser Ile Lys Arg Ser Ser Pro Gly Thr 245 250255 Cys Glu Thr Thr Gly Ser Pro Leu Met Leu Asp Glu Gly Asn Asp 260 265270 Asn Glu Val Tyr Phe Thr Tyr Ser Val Lys Phe Asn Glu Ser Ala 275 280285 Thr Ser Trp Ala Thr Arg Trp Asp Lys Tyr Leu His Val Tyr Asp 290 295300 Pro Ser Ile Gln Trp Phe Ser Leu Ile Asn Phe Ser Leu Val Val 305 310315 Val Leu Leu Ser Ser Val Val Ile His Ser Leu Leu Arg Ala Leu 320 325330 Lys Ser Asp Phe Ala Arg Tyr Asn Glu Leu Asn Leu Asp Asp Asp 335 340345 Phe Gln Glu Asp Ser Gly Trp Lys Leu Asn His Gly Asp Val Phe 350 355360 Arg Ser Pro Ser Gln Ser Leu Thr Leu Ser Ile Leu Val Gly Ser 365 370375 Gly Val Gln Leu Phe Leu Met Val Thr Cys Ser Ile Phe Phe Ala 380 385390 Ala Leu Gly Phe Leu Ser Pro Ser Ser Arg Gly Ser Leu Ala Thr 395 400405 Val Met Phe Ile Leu Tyr Ala Leu Phe Gly Phe Val Gly Ser Tyr 410 415420 Thr Ser Met Gly Ile Tyr Lys Phe Phe Asn Gly Pro Tyr Trp Lys 425 430435 Ala Asn Leu Ile Leu Thr Pro Leu Leu Val Pro Gly Ala Ile Leu 440 445450 Leu Ile Ile Ile Ala Leu Asn Phe Phe Leu Met Phe Val His Ser 455 460465 Ser Gly Val Ile Pro Ala Ser Thr Leu Phe Phe Met Val Phe Leu 470 475480 Trp Phe Leu Phe Ser Ile Pro Leu Ser Phe Ala Gly Ser Leu Ile 485 490495 Ala Arg Lys Arg Cys His Trp Asp Glu His Pro Thr Lys Thr Asn 500 505510 Gln Ile Ala Arg Gln Ile Pro Phe Gln Pro Trp Tyr Leu Lys Thr 515 520525 Ile Pro Ala Thr Leu Ile Ala Gly Ile Phe Pro Phe Gly Ser Ile 530 535540 Ala Val Glu Leu Tyr Phe Ile Tyr Thr Ser Leu Trp Phe Asn Lys 545 550555 Ile Phe Tyr Met Phe Gly Phe Leu Phe Phe Ser Phe Leu Leu Leu 560 565570 Thr Leu Thr Ser Ser Leu Val Thr Ile Leu Ile Thr Tyr His Ser 575 580585 Leu Cys Leu Glu Asn Trp Lys Trp Gln Trp Arg Gly Phe Ile Ile 590 595600 Gly Gly Ala Gly Cys Ala Leu Tyr Val Phe Ile His Ser Ile Leu 605 610615 Phe Thr Lys Phe Lys Leu Gly Gly Phe Thr Thr Ile Val Leu Tyr 620 625630 Val Gly Tyr Ser Ser Val Ile Ser Leu Leu Cys Cys Leu Val Thr 635 640645 Gly Ser Ile Gly Phe Ile Ser Ser Met Leu Phe Val Arg Lys Ile 650 655660 Tyr Ser Ser Ile Lys Val Asp 665

What is claimed is:
 1. An isolated cDNA, or the complement thereof,encoding a protein selected from: a) an amino acid sequence of SEQ IDNO:1; b) a variant having at least 85% identity to the amino acidsequence of SEQ ID NO:1; c) an antigenic epitope of SEQ ID NO:1; and d)a biologically active portion of SEQ ID NO:1.
 2. An isolated cDNA or thecomplement thereof selected from: a) a nucleic acid sequence of SEQ IDNO:3; b) a fragment of SEQ ID NO:3 selected from SEQ ID NOs:4-10; and c)a variant of SEQ ID NO:3 having at least 85% identity to the nucleicacid sequence of SEQ ID NO:3.
 3. The composition comprising the cDNA orthe complement of the cDNA of claim
 1. 4. A composition comprising thecDNA or the complement of the cDNA of claim 1 and a substrate.
 5. Aprobe comprising the cDNA or the complement of the cDNA of claim
 1. 6. Avector comprising the cDNA of claim
 1. 7. A host cell comprising thevector of claim
 6. 8. A method for producing a protein, the methodcomprising: a) culturing the host cell of claim 7 under conditions forprotein expression; and b) recovering the protein from the host cellculture.
 9. A transgenic cell line or organism comprising the vector ofclaim
 6. 10. A method for using a cDNA to detect the differentialexpression of a nucleic acid in a sample comprising: a) hybridizing theprobe of claim 5 to the nucleic acids, thereby forming hybridizationcomplexes; and b) comparing hybridization complex formation with astandard, wherein the comparison indicates the differential expressionof the cDNA in the sample.
 11. The method of claim 10 further comprisingamplifying the nucleic acids of the sample prior to hybridization. 12.The method of claim 10 wherein detection of differential expression ofthe cDNA is diagnostic of a thyroid follicular adenoma or thyroidlymphocytic thyroiditis.
 13. A method of using a cDNA to screen aplurality of molecules or compounds, the method comprising: a) combiningthe cDNA of claim 1 with a plurality of molecules or compounds underconditions to allow specific binding; and b) detecting specific binding,thereby identifying a molecule or compound which specifically binds thecDNA.
 14. The method of claim 13 wherein the molecules or compounds areselected from DNA molecules, RNA molecules, peptide nucleic acids,artificial chromosome constructions, peptides, transcription factors,repressors, and regulatory molecules.
 15. A purified protein selectedfrom: a) an amino acid sequence of SEQ ID NO:1; b) a variant of SEQ IDNO:1 having at least 85% identity to the amino acid sequence of SEQ IDNO:1; c) an antigenic epitope of SEQ ID NO:1; and d) a biologicallyactive portion of SEQ ID NO:1.
 16. A composition comprising the proteinof claim
 15. 17. A method for using a protein to screen a plurality ofmolecules or compounds to identify at least one ligand, the methodcomprising: a) combining the protein of claim 15 with the molecules orcompounds under conditions to allow specific binding; and b) detectingspecific binding, thereby identifying a ligand which specifically bindsthe protein.
 18. The method of claim 17 wherein the molecules orcompounds are selected from DNA molecules, RNA molecules, peptidenucleic acids, peptides, proteins, mimetics, agonists, antagonists,antibodies, immunoglobulins, inhibitors, and drugs.
 19. A method ofusing a mammalian protein to prepare and purify antibodies comprising:a) immunizing a animal with the protein of claim 15 under conditions toelicit an antibody response; b) isolating animal antibodies; c)attaching the protein to a substrate; d) contacting the substrate withisolated antibodies under conditions to allow specific binding to theprotein; e) dissociating the antibodies from the protein, therebyobtaining purified antibodies.
 20. An isolated antibody whichspecifically binds to a protein of claim
 15. 21. A diagnostic test for acondition or disease associated with the expression of VMP1 in abiological sample, the method comprising: a) combining the biologicalsample with an antibody of claim 20, under conditions suitable for theantibody to bind the polypeptide and form an antibody:polypeptidecomplex, and b) detecting the complex, wherein the presence of thecomplex correlates with the presence of the polypeptide in thebiological sample.
 22. The antibody of claim 11, wherein the antibodyis: a) a chimeric antibody, b) a single chain antibody, c) a Fabfragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 23. Acomposition comprising an antibody of claim 20 and an acceptableexcipient.
 24. A method of diagnosing a condition or disease associatedwith the expression of VMP1 in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 23. 25. Acomposition of claim 23, wherein the antibody is labeled.
 26. A methodof diagnosing a condition or disease associated with the expression ofVMP1 in a subject, comprising administering to said subject an effectiveamount of the composition of claim
 25. 27. A method of preparing apolyclonal antibody with the specificity of the antibody of claim 20,the method comprising: a) immunizing an animal with a polypeptideconsisting of an amino acid sequence of SEQ ID NO:1, or an immunogenicfragment thereof, under conditions to elicit an antibody response, b)isolating antibodies from said animal, and c) screening the isolatedantibodies with the polypeptide, thereby identifying a polyclonalantibody which binds specifically to a polypeptide comprising an aminoacid sequence of SEQ ID NO:1.
 28. A polyclonal antibody produced by amethod of claim
 27. 29. A composition comprising the polyclonal antibodyof claim 28 and a suitable carrier.
 30. A method of making a monoclonalantibody with the specificity of the antibody of claim 20, the methodcomprising: a) immunizing an animal with a polypeptide consisting of anamino acid sequence of SEQ ID NO:1, or an immunogenic fragment thereof,under conditions to elicit an antibody response, b) isolating antibodyproducing cells from the animal, c) fusing the antibody producing cellswith immortalized cells to form monoclonal antibody-producing hybridomacells, d) culturing the hybridoma cells, and e) isolating from theculture monoclonal antibody which binds specifically to a polypeptidecomprising an amino acid sequence of SEQ ID NO:1.
 31. A monoclonalantibody produced by a method of claim
 30. 32. A composition comprisingthe monoclonal antibody of claim 31 and a suitable carrier.
 33. Theantibody of claim 20, wherein the monoclonal antibody is produced byscreening a Fab expression library.
 34. The antibody of claim 20,wherein the antibody is produced by screening a recombinantimmunoglobulin library.
 35. A method of detecting a polypeptidecomprising an amino acid sequence of SEQ ID NO:1 in a sample, the methodcomprising: a) incubating the antibody of claim 20 with a sample underconditions to allow specific binding of the antibody and thepolypeptide, and b) detecting specific binding, wherein specific bindingindicates the presence of a polypeptide comprising an amino acidsequence of SEQ ID NO:1 in the sample.
 36. A method of purifying apolypeptide comprising an amino acid sequence of SEQ ID NO:1 from asample, the method comprising: a) incubating the antibody of claim 20with a sample under conditions to allow specific binding of the antibodyand the polypeptide, and b) separating the antibody from the sample andobtaining the purified polypeptide comprising an amino acid sequence ofSEQ ID NO:1.